CA1213545A

CA1213545A - Positive selection sorting of cells

Info

Publication number: CA1213545A
Application number: CA000433536A
Authority: CA
Inventors: Melvin S. Schindler; John F. Holland
Original assignee: Michigan State University MSU
Current assignee: Michigan State University MSU
Priority date: 1982-07-29
Filing date: 1983-07-29
Publication date: 1986-11-04
Also published as: US4624915A

Abstract

ABSTRACT OF THE DISCLOSURE
A method and apparatus for positive selection of viable cells based upon differing chemical or physical properties or dynamic processes using a focussed radiant energy beam, such as a laser beam (11) to kill unwanted cells or to isolate wanted cells is described. The apparatus includes microscope means (14) and cell detection means (27) for identifying the cells to be killed or saved and attenuator means (12) for modifying the beam to prevent destruction of cells to be saved.

Description

3S4~;
BACKGROUND OF THE INVENTION
The present inventlon relates to an apparatus and a method for sorting a population of livinq cells based upon differing chemical or physical properties or dynamic process capabilltles. The method uses a radiant energy beam (11, 15) for destroying unwanted cells and/or circum-scribing around wanted cells in the population Prior Art In ~eneral in the prior art, a central feature of the chemical analysis of complex mixtures is the isolating and purification of specific compounds. These purified homogeneous components are subsequently characterized with regard to their physical properties. In the past, for cell biochemistry this implied the isolation from the cell of an enzyme or receptor protein by gel chromatography with the attendant destruction of the cell.
With the advent of newer culture techniques and defined media, it has become possible to grow all types of cell lines in culture. This has provided for the possibili_y of growing sufficient quantities of cells to do biochemical analysis on cell and membrane components. A major problem in this approach, however, is the large variability of cell surface structure leading to altered function within a relatively specific family or population of cells. For example, T-cells of the immune system have sub-populations of cells with altered properties and function. These obser-~a-tions have stimulated researchers ~-2~
to develop methodologies and instrumentation for the purpose of separating the cells into di~tinct sub-population~ on the basis of a defined characterization in a direct analogy to the conceptual approache~ of protein separation and purification mentioned above. Mixed polymer systems have been employed to separate cells based on surface properties, cell electrophoresis ha~ been used to separate cell3 by surface charge, and solid phase lectins and antibodies have been exploited to differentiate between altered ~tructural determinants in membrane proteins for cell separationO
Recently, fluorescence measuring cell sorters have become commercially available. The great advantage of the method and apparatus of these devices is that they efficiently separate a large population of cells based on differences in bonnd fluorescent probes (usually an antibody or lectin containing a covalently attached fluorophore). Specific selection of parameters are established in these prior art instruments. Cells are sorted into separate tubes based on fluorescent or other light interactive behavioral differences. Sorted cells can be run through the system a number of times to provide further enrichment. In this manner, approximately l x 107 cells, an amount sufficient for characterization, could be sorted in l to 2 hours.
The fluorescence activated cell sorter (FACS
Systems, Becton Dickinson of Sunnyvale, California U.S~A.
and Coulter Electronic, Inc. of Hialeah, Florida) are computer centered, single laser units capable of analyzing and separating individual cells on the basis of fluorescence, size and viability. The electronic and recording measurements can be made with or without sorting.
Sorting is accomplished by electrostatic induction on individual cells in a flow system. The laser is used as a light source for the fluorescence. The cytofluorograph system (Ortho Diagnostic Systems, Inc. of Westwood, Ma~sachusetts) utilizes two laser beams in the same manner and separates again by electrostatic induction ~3~ ~2~35~
based upon m~asurement~ of ~ize, fluorescence and/or refraction indices.
Although this prior art methodology and apparatus represent a major technological breakthrough with regard ~o obtaining a homogeneous population of cells based upon chemical properties, its usefulne ~ is limited in the following ways:
(1) since all are flow through systems, they are su~ceptible to the pump and clogging problems observed in any fluid system5 ~ 2) maintaining sterile conditions through long lengths of plastic tubing through which the cells must pass is very di~ficult;
(3) the buffeting and collisions encountered by cells flowing through the sorter can have deleterious effects on cell m~embranes, causing unknown cellular responses, in some cases cell death;
(4) cell differentiation in most instances is determined only by structural marker~ attached to the cells which does not take advantage of the ability to isolate and separate cells based on altered membrane properties such as the rotational and translational diffusion differences of cell surface components. These parameters have been shown to be important in cell stimulatory events, e.g.-hormones and growth factors;
(5) a major drawback in flow cytometry sorter~
is the necessity to use cells in suspension for separation.
This preclude~ the use of this technique for the sorting of cells growing in culture mounted on a plate or surface.
The properties of cells can change dramatically with shape;
it is therefore essential to sort them a~ close to their normal shape as possibLe. In addition, there ha~ been a strong correlation made between c~ll shape and ultimate function~ both of which are affected by the surface on which the cell~ grow. Clearly, it would be desirable to separate the cells while growing on specieic surfaces.
This is not possible with the commercial unit~.

The prior art in Xoppel, D. E. et al Biophysics Journal 28A281-291 (1979) has described the use of lasers for irradlating cells mounted on a plate with light energy sufficient to photobleach a spot on a surface of the cell and then to follow the progress of the migration of fluorescently labeled molecules back into the bleached area.
In the Koppel et al apparatus, occasionally the cells can be accidentally killed by the laser beam;
however, the apparatus is adapted only for practice of the bleaching method. No provision is made for providing a supply of nutrient medium to the cells and thus there is no means for removing the enzymes and other cell components left in the event the cells are killed. Such components seriously affect the remaining living cells. The Koppel apparatus has never been used in a method for cell sorting.
Other prior art practices include cell or cellular component destruction by laser energy, such as by Higgins, et al., J. Neurosc. Meth., 3, 1, 1980; Bessis in Advances in Biological and Medical Physics, Academic Press, New York;
1970; ~osley, et al., in Proc. Natl. Acad. Sci. 78, 9, 1981;
Lepok, et al., Biochem. Biophys. Res. Comm. 91, 3, 1979;
and Berns in Jour. Cell Biol. 75, 3, 1975.
All oE these u-tilize the destructive power of ultravlolet or coherent radiation for the purpose of destroying selected targets, not for the purpose of cell progen~ selection in a life sustaining environment. The lcm/,~

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application of this prior art involves ablation studies, cell suicide studies (negative selection), Organelle micro-surgery, therapeu~ic cell destruction and other areas not relating to cell sorting.
According to a first method aspect of the inven-tion there is provided a method for sorting anchorage dependent cells by selective destruction of unwanted cells in situ and preserving wanted cells in situ comprising the steps of: culturing a heterogeneous population of anchor-age dependent cells on a surface; identifying wanted from unwanted cells on the surface by focussing radiant energy beam means upon the cells, the beam means eliciting a radiative response depending upon selected physical, chemical and dynamic properties of the cells in situ at a first energy intensity for discriminating between wanted and unwanted cells in situ and automatically subjecting the unwanted cells to the beam at a second energy intensity lethal to the unwanted cells thereby preserving in situ the wanted cells.
~ccording to a further method aspect of the inven-tion there is provided a method for sorting of a hetero-geneous population of anchorage dependent living cells to achieve separate segregated populations of living cells which process includes: providing a film support for the attachment of a heterogeneous population of cells on an underlying surEace; automatically apply.ing a radiant energy beam at a non-lethal intensity to all of the cells on ~he film support; automatically identifying different of the lcm/~

-5a- ~3~5 cells by observing physical, chemical and dynamic pxoperties of the cells as elicited by the radiant energy beam in situ on the film support surface; automatically applying the focussed radiant energy beam at a second intensity to circumscribe selected of the cells in situ on the film support surface by thermal fusion of the film to the under-lying surface thereby enabliny isolation of one group of the cells from others of the cells on the film support surface; automatically coordinating movement of the support for the cells with the identifying of the cells at the first mentioned intensity of the radiant energy beam where-by the presentation of the cells, the identification of selected cells and the application of the isolating second intensity of the focussed radiant energy beam to the cells is automatic; and removing the others of the cells from the one group of the cells by removal of the film from the underlying support leaving the one group of cells on the underlying surface.
According to a first apparatus aspect of the inven-tion there is provided an apparatus for automatic sorting of anchorage dependent cells by destruction of unwanted cells in situ, the combination comprising: a cell support surface;
a focussed radiant energy beam selectively directed in a path intersecting the cell support surface, the beam and the surface relatively movable in respect to each other to access the beam to all cells attached to the surface;
discriminating means which observes the response of the cells to the beam at a selected non-lethal first energy f..~ ~

lcm/~

-Sb- ~2~3~5 intensity and determines wanted from unwanted of the cells;
adjusting means selectively altering the intensity of the beam to deliver a selected lethal energy intensity to destroy unwanted of the cells in situ on the surface as established by the discriminating means; and automatic means to coordinate and direct selected movement of the beam in relationship to the surface to impact the beam with all of the cells on the surface in a motion coordinated with the . discriminating means at non-lethal first intensity of the beam and then automatically adjusting the beam at a second energy intensity lethal to selected unwanted of the identi-fied cells.
According to a second apparatus aspect of the invention there is provided an apparatus for sorting anchored cells by destruction of unwanted cells, the combination comprising: a cell support surface having connections for nutritional, thermal and chemical adjustment of cell environ-ment; a focussed radiant energy beam having at least two energy intensities and directed in a path intersecting the cell support surface and the beam and the surface relatively movable in respect to each other to access the beam to all cells attached to the surface; cell discriminating means in an aligned relation with the beam and intersecting the cell surface in congruence with the beam and being connected to the beam to provide a first and non~lethal enexgy intensity for selected recognition of wanted and unwanted cells and the discriminating means selecting a second and lethal energy ! lcm/~

-5c~ S~ S

intensity for the beam at other selected recognition criteria; adjusting means providing the feedback control connection responsive to the discriminating means and selective of the energy intensity of the beam; and auto matic coo.rdinating means in control of movement of the beam in relation to the cell support surface in traversing the entire of the surface, automatically responsive to recogni-tion by the discriminating means and shifting of the beam from non lethal to lethal intensity by manipulation of the adjusting means, and selectively sustaining the environment of the ce.lls on the support surface in preservation of cell life support conditions and the flushing of debris from the support surface during and after lethal exposure of unwanted cells on the support surface to the beam at lethal intensity.
According -to a further apparatus aspect of the invention there is provided an apparatus for sorting a heterogeneous population of anchorage dependent living cells in culture in situ into a segregated population based upon chemical, physical and dynamic properties of the cells comprising: shielded automatic optical means; a cell support surface film providing a focal stage for the optical means on an underlying surface; a focussed radiant energy beam means automatically directing a beam at a selected first energy intensity and a selected second energy intensity in a path determined by and in focal reyistry wi.th the optical means focussed upon the cells on the film support surface, the beam and the surface in relative automa-tic movement to expose all of the cells to the beam; discrimination means ¦ lcm/~

-5d- 1213S45 automatically identifying individual cells upon the cell support surface in accord with selected chemcial, physical, or dynamic cell properties as elicited by the beam at first energy intensity and determined through the optical means;
and automatic control means connected to the radiant energy beam providing selected plural energy levels of the beam to the film support for selected irradiation, circum-scription and isolation of selected cells on the film support surface as indicated by the discriminating means and isolation of selected encountered cells by a circumscribing application of a selected second energy intensity of the beam.

lcm/~, ~ ~

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-I~ the Drawin~
Figure 1 is a block diagram of the preferred po~itive selection cell sorting apparatu~ used in the me~hod of the pre ent invention.
Figure 2 is a ~chematic view of the laser (10), beam attenuator (12) and microscope m~a~ (14) in the preferred apparatus of the pre~ent inventîon.
Figure 3 is a block diagram showing the interface between the detection means (27, 106) and the computer (101) in the preferred apparatus.
Figure 4 is a schematic diagram showlng preferred means for supplying a continuous flow of nutrient medlum to cells on a plate (17) adapted to contain the medium.
Flgure 5 is an isometric exploded view of the plate (17) sho~n in Figure 2 showing an x-y moveable stage (16) for the plate ~17).
Figure 6 i~ a front cross-sectional view of the plate (17) shown in Figure 5.
Figures 6a, 6b and 6c show one slide (311a) with a film (3121 mounted on it such that wanted cells can be circum~cribed by the beam (15) and unwanted cells removed with the film (312) except for discs (313) with the wanted cells.
General-Description The present invention relate~ to an apparatus for sorting a heterogeneous population of living cells into a segregated population based upon chemical or physical properties or dynamic processes by selection for wanted cells which comprises:
(a) microqcope means ~14) with an objective (15a) for scanning a heterogeneous population of cells fixed on a plate ~17, 3111 or on a film (312) removably mounted on the plate positioned adjacent the objective on a coordinate scanning or an individual cell-by-cell scanning ba~is, wherein the plate or film on the plate i9 adapted to contain a flowing liquid growth medium for the cells;
,.

_7~ 3 ~ ~ 5 (b) detec~ion means (27~ 106) ~or di~tingui~hing an individual cell ba~ed upon a particular chemlcal or physical property or dynamic processe3 a~
determined by the detectLon mean~ through the objective;
(c) focus~ed radiant energy beam (11, 15) generating mean~ (10, 11, 12, 13, 15a) as~ociated with the microscope means ~uch that the beam can ~e focussed in a path through the objective at the plate and at or around a~
individual cell or series of cells on the plate; and (d) controlled interruptio~ means ~12) for selectively attenuating the beam, wherein the beam can irradiate or circum~cribe individual cells on the plate or on the film on the plate when the interruption means i~
removed from the path.
The present invention further relate~ to a preferred apparatus for sorting a heterogeneous population of living cell3 into a segregated population based upon physical or chemical properties~or dynamic processes which comprises:
~a) microscope means (14) with an objective for viewing a heterogeneous population of cells, which are fixed on a plate (17, 311) or on a film (312) removably mounted on the plate, with the plate positioned adjacent the objective on a coordinate scanning or an individual cell-by cell scanning basis, wherein the plate or Eilm on the plate i~ adapted to contain a flowing liquid growth medium for the cells;
(b) drive means (100) for moving the plate beneath the objective or for moving the objective or beam across the plate in an x-y coordinate plane or for both such that the obje~tive scan~ and views each of the cell~
in the heterogeneous population;
(c) detection means (27, 106) for distingui~hing an individual cell based upon a particular chemical or physical property or dynamic proce~ses determined by the detection means through the objectives (d) focussed radiant energy beam (11, 15) generating means (10, 11, 13, 15a) with lenses ~22) and -8- ~z~35~
mirrors (13a, 13b, 13c, 13d, 13e) po~itioned for directing the beam in a path through the objective and at or around an individual cel~ or a series o~ cells viewed by the objective;
(e~ attenuator means (32, 33, 34, 35, 3~) move~ble into the beam for selectively attenuating the beam;
(f) controlled means (32, 33, 34, 35, 36) for ~electively providing the attenuator mean~ into th~ path of the beam, wherein the detection mean~ distinguishe~ cell~
to be saved-when the attenuator means is positioned between the objective and the beam by the controlled mean~ and wherein the attenuator means is removed from the path of the beam by the controlled means in order to use the beam to select for unwanted or wanted cells by killing unwanted cells or by fusing the film onto the plate supporting the cell~ around the wanted cells with the beam or by cutting around a ilm removably adhered~to the plate and then removing the film with the unwanted cells fixed to the film.
The present invention also relates to a method for selecting for wanted metabolizing living cells which comprises:
(a) providing the cells fixed on a plate (17, 311) or a plastic film (312) mounted on the plate beneath a microscope means (14) with an objective (15a) wherein the objective view~ the cells on an indi.vidual cell-by-cell or series of cell~ basis and wherein the plate contains a flowing liquid growth medium for the cells; and (b) selectively attenuating (28, 29, 30, 31) the beam (11) such that cells to be saved are not irradiated with the unattenuated beam and such that the cells to be saved are selected based upon a light response to the attenuated beam; and (c) selectively irradiating at or around the individual cells or 3eries of cells with a focussed radiant energy beam (11, 15) through the objective (15a) of the microscope means with ~ufficient energy to kill unwanted -9~ 3 ~ ~ ~
oell3 on the plate or to ~ircum~crîbe wanted cell~ on the film so that the wanted cell~ are separated with a portion of the film and re~ain on the plate and the u~wanted cells are removed.
Finally the pre~ent inventio~ relate~ to a preferred method ~or ~orting a heterogeneou~ population of cell~ into a segregated populatio~ based upon phy~ical or chemical propertie~ or process capabilities by selectively killing unwanted cell~ or by retaining wanted cell~ which comprises:
(a) providing a cell sorting apparatus including a microscope ~14) with an objective (15a) for viewing a heterogeneous population of cells fixed on a plate (17, 311) or a film (312) mounted on the plate positioned adjacent the objective on a coordinate scanning or an individual cell-by-cell scanning basis wherein the plate or the film on the plat~ contain~ a flowing liquid growth medium for the cell~;
drive means (100) for moving the plate and container beneath the objective or for moving the objective or beam acros~ the plate in an x-y coordinate plane or for both such that the objective scans and views each of the cell3 in the heterogeneous population;
detection mean~ (27, 106) for distinguishing an individual cell based upon a particular chemical or physical property or dynamic process determined by the detection means through the objective;
focussed radiant energy beam (11, 15) generating mean~ (10, 11, 13, 15a) with lenses (22) and mirror~ (13a, 13b, 13c, 13d) po~itioned for directing the beam in a path through the objective at or around an individual cell or a series of cells viewed by the objective:
attenuator means (28, 29, 30, 31) moveable into the path of the coherent light beam for selectively attenuating the beam;
controlled mean~ (32, 33, 34, 35) for selectively providing the attenuator means into the path of the beam, wherein the detection means distingui~es cell~

-la ~21 359~5 to be saved when the attenuation means ls po~itioned between the sbjective and the beam and wherein the attenuator means i~ removed from the path of the beam by the controlled mean~ in order to use the beam to select or unwanted or wanted cell~ by killing unwanted cells or to circumscribe the wanted cells by fusing the ilm onto the plate supporting the cells around the wanted cell~ with the beam or by cuttins around a film removably adhered to the plate with the beam and then removing the film with the unwanted cells fixed to the film;
(b) ortlng the cells by ~electively allowing the beam to kill unwanted cells or to circum~cribe the wanted cells with the beam by removal of the attenuator mean~ from the beam.
SPecific DescriPtion Optical System Referring to Figure3 1 and 2 a focussed radiant energy beam generating means, ~ch as a laser 10, generates a beam 11. The laser 13 is preferably of the argon type and capable of generating light in wavelengths between about 300 and 560 nanometers. The beam 11 is filtered by beam attenuation means 12 provided in the path of the beam 11. Beam deflection mean~ 13, such as mirrors, 13a, 13b, 13c, 13d and 13e ~hown in Figure 2, are used to provide the beam 11 inside a microscope 14. A focussed beam 15 is provided by an objective 15a. The focussed beam 15 mea~ures about 1 micron in diameter compared to cells of a much greater dimension, for example 40 microns in diameter with nucleus of about 10 microns in diameter.
A microscope stage 16 supports plate 17 for supporting the immobilized cell~ ~not shown). The stage 16 i~ veable in an x and y plane perpendicular to the focu~sed beam 15 a~ discussed in connection with Figure 5.
The mirrors 13c and 13d are moveable by motor~ 18 and 18a ~o as to provide ~canning of the plate 17 in the x and the y plane by the focu ~ed beam 15. I the stage 16 moves a~
described hereinafter, then the motor~ 18 and l~a are unne.cessary. Diaphragms 19 and 20 can be u~ed to regulate .

35~i tha beam as can shutter 21. Lens 22 contributes to movemen~ of the beam 15 in the x or y coordinate without distortion when mirrors 13c and 13d are moved.
Mirror 13e i~ a dichromatic mirror ~uch that emitted light 23 from the plate 17 pa~es through the mlrror 13e. A barrier filter 24 allow~ only selected luminescence to pas~. A mirror or grating element 25 i~
po~ltioned in the path of the reflected liqht 23. A
shutter 26 allow~ instantaneous passage of light to a photo multiplier tube 27.
The ba~ic elements of the laser microscope apparatus ~without the attenuator mean~ 12 or the unique moveable stage 16 as di~cussed hereinafter~ are described in detail in the Koppel article discusQed previously. The lS laser is available from Lexel, Inc., Sunnyvale, California;
the micro~cope is available from Leitz, Inc., Rockleigh, New Jersey and the moveable mirrors 13c and 13d are available from General Scanning~ Inc., Waterstown, Massachusetts.
The beam attenuation means 12 includes filters 28, 29, 30 and 31 which are moveable into and out of the beam 11 path by means of pneumatic actuators 32, 33, 34 and 35. ~ilter 28 is moved to position 2Ba out of the beam 11 path by the actuator 32 a shown by the dotted lines. The remaining filters 29 to 31 are moved in the same manner.
Valves V control the movement of the actuators 32 to 35.
Pneumatic lines 36 provide air under pressure to valve~ V.
The microscope includes a conventlonal binocular viewing means 37, support 38 and a manual adjustment knob 39.
Electronic_Confiquration Referring to Figures 1 and 3, the electronic elements of the present invention are illustrated. The stage 16 ls provided with electrically actuatable controls 100, as de~cribed more fully hereinafter, to provide movement o~ the ~tage 16 in the x-y plane perpendicular to the beam 11. Alternatively the motors 18 and 18a for mlrrors 13c and 13d can be electrically moveable. The -12- ~2~35~
stage 16 movement i controlled by a computer 101 driven by a program stored on a disc(s) 102. The computer 101 can include video display 103 and recor~er (not ~hown) connected to terminal 104. The microscope 14 functions a~
a fluore~cence collection unit 105 from the ~age 16. The photomultipliex tube 21 function for fluorescence detection 106 and includes mean~ 106a for counting and/or measuring the fluorescence which i9 recorded by the computer 101. Thu~ the computer 100 controls stage 16 movement and records the presence or absence of fluorescence on the plate 17. For ~lower operation a system without the computer 100 could be used; however with much les~ precision and the speed would be prohibitively slow. The lens 15a provides beam 11 Eocussing, the mirrors 13a to 13e provide beam direction and the beam attenuator 12 provides beam attenuation as previously di cu~sed~
In Figure 3, the detector or photomultiplier tube 27 is coupled to the comput,er 101 by means of switch 107 to either: (a) an amplifier discriminator circuit 108 manufactured by Precision Products, Inc., Groton, Connecticut and counter lOg through interface 110 to the computer lOi; or (b) to a rate measuring circuit using the analog amplifier 111 and a 12 bit analog to digital converter 112. The digital outp~t of this unit is fed to the computer 101 through ~he interface 110. All of thi~ is state of the art.
Cell Environmental Control Figures 4, 5 and 6 show the plate 17 adapted for cell environmental control on stage 16. Container 200 is fed through line 201 with a mixture of cell growth media from containers 202 and 203 controlled by valves V in lines 204 and 205. Container 206 feeds necessary gases to container 200 through valve V in line 207. Pump P supplies the mixed media in container 200 and ga~es via line 208 to plate 17 as shown more fully in Figures 5 and 6. Line 209 release~ spent media to a drain or waste container 210.
Monitoring of media properties i~ accompli~hed ~y tran~ducers ~ensitive to temperature, carbon di~xide, - ~Z~35~L~

oxygen and hydrogen ion actLvity. These transducer~ are connected to unit 211.
The stage 16 include~ motor 300 for movement in the x direction and by motor 301 for movement i~ the y direction. The movement is accomplished by screw shafts 300a and 301a. Attached by plate3 302 and 303 to a first moveable holder 304 whicb slides in the x direction on base 305 and to a ~econd moveable holder 306 which slides in the y direction on first holder 304. Such a stage is available from Ealing located in South Natick, Massachusett~.
The second holder 306 i5 provided with a recess 306a adapted to receive the base 307a of a support 307.
The support 307 has spaced apart recesses 307a and 307b and 307c adapted to receive a container 308 with inlet conduit 309 and outlet conduit 310 to be connected to lines 208 and 209 as shown in Figure 4. The container 308 includes integral pedestals 308a, 308b and 308c which support glass slide3 311a, 311b and 311c which support the growing cells.
The support 307 includes clip 312 for holding glass cover 313 in position on support 307 to completely cover the plate 308 when the cell~ are not being viewed by the objective 15a. During viewing of the cells on the slides 311a to 311c by the objective, the cover 313 i~ removed;
how~ver, the media is still allowed to flow across the con~ainer 308.
As shown in Figures 6a to 6c, a variation of the method of the present invention utilizes a thin film 312 mounted on the slide 311a which supports the cells so that the beam 15 cu~s or fuses the film 312 to the slide 311a around the cells for removal of unwanted cells with the film 312. One slide 311a with the thin film 312 as shown in Figure 3a. The film 312 i~ generally between about 5 and 200 micron~ thick is in contact with the slide 311a and the cells yrow on the opposite side of the film 312. The fil~l is preferably composed of a thermopla~tic material such as polyvinyl chloride. The ilm 312 can also be removably bonded to the slide 311a with an adhesive so that the beam 15 can cut around the wanted cells and the -14~ 3~3~ ~
unwan ed cells ar~ remcved with the film 312 from the ~lide 311a. Alternatively the beam 15a ca~ fu~e portions of the film 312 to the ~lide 311a by beam lS ~elding. In either ca~e the unwanted cell3 and film 312 can be stripped from the plate to leave behind di~c~ 313 ~upporting the ~anted cell~ from film 312 fused onto the slide 311a.
~ he improved method is valuable in sorting or a few wanted cells in a large number of cells, since it produces little celL debris which can aEfect the wanted cells. Where there are fewer unwanted cells in the cell population, the unwanted cell killing method is preferred.
Light ab~orbing material9 can be provided on the ~lide 311a alone or beneath the film 312. This facilitate~
cell killing or cutting or fusing of the fllm 312 u~ing the beam 15.
It will be appreciated that the method involving circumscribing wanted cells by cutting or fuqing with the beam 15 can be combined with th~ method involving killing of the unwanted cell~. There is no particular advantage in using the combina~ion for one sorting procedure because of the concern for cell debri~ which affects wanted cells in the culture medium.
Operation The present invention uses quantitative techniques for the characterization of diffusion in bîological membrane~ which has made it po~3ible to employ laser technology to develop a scanning system capable of selecting a specffic sub-population of cells ba~ed on a variety of structural and/or physical parameters. The cells do not move because they are supported on slides 311a to 311c or Eilm 312 and can be maintained in a state that Ls maximally advantageou~ to the maintenance of cell viability~ The central idea of the approach is that cells wLth the desired property are spared from a pul~e oE high intensity laser beam 15a capable of destroying them or are circumscribed when they are on the film 312. In this manner, a high level of cell enrichment for the defined characteristic i~ obtained. A 3ignificant feature of this Z~3S9~i ~y3tem is that cells cultured on slides 311 or film 312 may be used for enrichment. This advantage greatly increases the ultimate biological relevance of any information obtained by chemical analysi~ of the resulting sorted cell~.
The intensity of the beam 11 i~ controlled by one or more neutral density filters 28 to 31 in~erted or removed from the beam 11 path by air-driven solenoids 3~ to 35 that are computer 101 controlled preferably in sixteen ~16) di~crete levels of attenuation~ Alternate devices to effect beam attenuation can be acousto-optical and/or electro-optical. A series of diaphragms and mirror~ 13a to 13e then guide the beam into the back of the microscope 14 capable o epi-fluore~cence illuminatio~. The microscope lS objective 15a adjust~ the final focus of the laser beam 15 into the plane of the cells. The dichroic mirror 13e reflects the laser beam 11 down through the microscope 14 objective while allowing sample luminescence to be transmitted up to the photomultiplier tube 27 located in a thermoelectrically cooled hou~ing 27a. The electronic shutter 26 placed in front of the tube 27 acts to protect the tube 27 from high levels of irradiation. The signal from the tube 27 is fed to a computer 101 for processing and output.
Tha essential feature of positive cell selection is the maintenance of optimal growth condition~ during the sorting cycle. The microflow container 308 provides a controlled environment for cells which can be further controlled by the computer 101 (not shown). This container 308 is preferably metallic and provides a thermal reservoir for temperature control, flow selection and regulating valves for media flow and ga~ mixing by the system shown in Figure 4~ This container 308 also provides or time dependent alterations, e.g. discrete growth substance addition, timed light exposure, etc. Sterility of growing cultures i~ maintained by the flow properties of the container 308. For short working distance the objective 15a is sterilized before use and is positioned in the ` -16- ~ ~13~
growth medlum. For long workiny distance objectives, immersion 1~ not necss~ary. Cell3 are grown in container 308 preferably with about 0~8 hunared forty-four mm2 area continuou31y bathed by the flowing media.
For the purpose of cell sorting, three operational mode~ can be utilizad. Cell sorting on the ba~is of a fluore~cence marker recognition; cell sortin~ on the ba~i~ of a mea~ured physical property; and cell ~orting on the basi~ of a measured dynamic process (tima dependent) characteri~tiC-The selection occur~ as follow~. The cell~ are grown on slides 311a to 311c which can have the film 312 on it. The edges of these slides 311a to 311c tor ~ilm 312 are coated with a strong fluorophore (not shown). A low intensity scout beam 15 traverses an ~x) coordinate of the field. On its return on the same coordinate, the high intensity beam 15 i9 turned on to kill unwanted cells or circumscribe wanted cells on the film 312. However, if luminescencP were detected during the scout beam traverse, the beam 15 will be attenuated on its return pas~. The positive detection of the di~ferentiating signal by the tube 27 ensures the survival of the selected cells.
Detection of the edge fluorophore on slides 311a to 311c serves as the boundaries for the scan field of the laser beam 15. The operator need only set the attenuated scout beam 15 on the plate 17. From this point, scanning of the total field i5 automatic and controlled by the computer 101 .
The positive cell 3election can be based upon 30 measurements of physical properties such as endogenous fluorescence and de-polari~ation as well as marker recognition. For more sophisticated application~, actual short e~perlments can be perormed af ter which ~election may be based upon membrane parameters such as rotational and/or tran~lational differences and microviscosity. In all cases, these proce~ses result in the i~olation of a viable ~ub-population o cells or functional clones that have undergone selection without mechanical perturbations.

-17~ 3~
~ he instrumentation for the~e types of measurement~ can be divided into three ~3) groups which are modular in the progression from the ~implest to the more complex.
Type I - Thiq apparatus i de~igned to provide selection ba~ed upon the cell's affinity for an internally or externally directed 3ite specific probe that is fluore~cently labeled, e.g.~antihodie3, lectins, DNA-RNA
intercalating dye~. Th~a ba,3ic component~ for this in~trumen~ are a tunable argon or argon-krypton laser 10, directing optics 13a to 13e and shutter 21, a microscope 14, a two-dimensional tran~lating stage 16 and photon or rate counter 106a. The instrument is controlled by the computer 101.
TyPe II - The addition of inciden~ and excitation polarizers and a monochrometer (not shown) in the emission beam before the photomultiplier tube 27 extends the capability of a Type I instrument to select on the basi~ of the polarization value of various fluorescently depolarizable dyes, e.g.-DPH, and also provide the researcher with the means to select cells that have altered fluorescence emission profiles for cell in~sorporated or adsorbed dyes. Selection i5 now based on actual changes in the physical state of the cell rather than purely as a function of the presence or absencf~a of a structural component.
~Ye~ Since alterations in the membrane dynamics of cell3 have been demonstrated to be intimately involved in cellular activity, the type III instrument is designed to make it~ selection on the basis of a real time experiment evaluating lateraI and/or rotational diffusion. Thi~ type of selection could not be performed on the flow separa'~ion system of the prior art due to the necessity for time re301ved measurements. Becau~e cell~ are plated and malntained in a viable environment, long term selections are fea,3ible based on individual cellular experimentation.
This type of instrumentation differs from Type II primarily in the computer 101 sotware and data processing power.

Definable Parameters of O~eration Operator control i~ attalned by keystroke command. In addition to initiating and terminating a sorting process, the following paramete~s can be ~cheduled under program control:
a) two-dimensional ~tage 16 movement with step magnitude and frequency control.
b) inten~ity of analytical (scout) and destroy or fuse beam~ 11.
c) threshold beam 11 inten~ity level.
d) mode selection - marker or dynamic.
e) thre~hold for positive response.
f) single Eield scan or continuou~ mode of operation.
g) method selection - cell destruction or cell isolation.
h) beam path radius for cell isolation.
i) control of all stage 16 operating function3:
i) temperature ii) media mixing and flow iii) gas injection iv) growth control additives v) light exposure vi) auxiliary time dependent parameter~.
Sorting analysis is now a major research technique in several disciplines, including cancer immunology, hematology, cell cycle analysis, pathology, biochemistry, general immunology and quantitative cytology.
New areas of intere~t for these methods are continuously emerging in cell biology, pharmacology, and toxicology, microbiology and cytogenetics. Diseases now under investigation with cell ~orting technology are leukemia, lymphoma, and other cancer~, infectious dlseases, auto immune diseases, and genetic di30rders. The apparatus of the present invention makes reliable cell selection possi~le for these investigations.

.,,

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for sorting anchorage dependent cells by selective destruction of unwanted cells in situ and pre-serving wanted cells in situ comprising the steps of:
culturing a heterogeneous population of anchorage dependent cells on a surface;
identifying wanted from unwanted cells on said surface by focussing radiant energy beam means upon said cells, said beam means eliciting a radiative response depend-ing upon selected physical, chemical and dynamic properties of said cells in situ at a first energy intensity for discriminating between wanted and unwanted cells in situ and automatically subjecting said unwanted cells to said beam at a second energy intensity lethal to said unwanted cells thereby preserving in situ said wanted cells.

2. A method for sorting of a heterogeneous population of anchorage dependent living cells to achieve separate segregated populations of living cells which process includes:
(a) providing a film support for the attachment of a heterogeneous population of cells on an underlying surface;
(b) automatically applying a radiant energy beam at a non-lethal intensity to all of said cells on said film support;
(c) automatically identifying different of said cells by observing physical, chemical and dynamic properties of said cells as elicited by said radiant energy beam in situ on said film support surface;
(d) automatically applying said focussed radiant energy beam at a second intensity to circumscribe selected of said cells in situ on said film support surface by thermal fusion of said film to said underlying surface thereby enabling isolation of one group of said cells from others of said cells on said film support surface, (e) automatically coordinating movement of said support for said cells with said identifying of said cells at said first mentioned intensity of said radiant energy beam whereby the presentation of said cells, the identification of selected cells and the application of the isolating second intensity of said focussed radiant energy beam to said cells is automatic; and (f) removing said others of said cells from said one group of said cells by removal of said film from said underlying support leaving said one group of cells on said underlying surface.

3. An apparatus for automatic sorting of anchorage dependent cells by destruction of unwanted cells in situ, the combination comprising:
a cell support surface;
a focussed radiant energy beam selectively directed in a path intersecting said cell support surface, said beam and said surface relatively movable in respect to each other to access said beam to all cells attached to said surface;

discriminating means which observes the response of said cells to said beam at a selected non-lethal first energy intensity and determines wanted from unwanted of said cells;
adjusting means selectively altering the intensity of said beam to deliver a selected lethal energy intensity to destroy unwanted of said cells in situ on said surface as established by said discriminating means; and automatic means to coordinate and direct selected movement of said beam in relationship to said surface to impact said beam with all of said cells on said surface in a motion coordinated with said discriminating means at non-lethal first intensity of said beam and then automatically adjusting said beam at a second energy intensity lethal to selected unwanted of said identified cells.

4. The apparatus of claim 3 in which the cell support surface is movable in respect to said radiant energy beam and wherein said cell support is operably attached to environmental support means for life support of said cells on said support surface, and wherein movement and environment is controlled by said automatic means.

5. The apparatus of claim 3 said discriminating means being a shielded photomultiplier tube observing said cells and sensing the response of said cells to coordinate with the function of said automatic coordinating means.

6. The apparatus of claim 3 in which said automatic coordinating means to identify, manipulate, locate and destroy unwanted cells in situ and preserve wanted cells is computer directed.

7. The apparatus of claim 6 in which said automatic coordinating means is connected controllably to cell environment and flushing means in preservation of life support for said cells on said cell surface and in avoidance of residue from destroyed cells contaminating remaining wanted cells.

8. An apparatus for sorting anchored cells by destruction of unwanted cells, the combination comprising:
a cell support surface having connections for nutritional, thermal and chemical adjustment of cell environment;
a focussed radiant energy beam having at least two energy intensities and directed in a path intersecting said cell support surface and said beam and said surface relatively movable in respect to each other to access said beam to all cells attached to said surface;
cell discriminating means in an aligned relation with said beam and intersecting said cell surface in con-gruence with said beam and being connected to said beam to provide a first and non-lethal energy intensity for selected recognition of wanted and unwanted cells and said discrimin-ating means selecting a second and lethal energy intensity for said beam at other selected recognition criteria;
adjusting means providing the feedback control connection responsive to said discriminating means and selective of the energy intensity of said beam; and automatic coordinating means in control of movement of said beam in relation to said cell support surface in traversing the entire of said surface, automatically responsive to recognition by said discriminating means and shifting of said beam from non-lethal to lethal intensity by manipulation of said adjusting means, and selectively sustaining the environment of said cells on said support surface in preservation of cell life support conditions and the flushing of debris from said support surface during and after lethal exposure of unwanted cells on said support surface to said beam at lethal intensity.

9. An apparatus for sorting a heterogeneous population of anchorage dependent living cells in culture in situ into a segregated population based upon chemical, physical and dynamic properties of said cells comprising:
(a) shielded automatic optical means;
(b) a cell support surface film providing a focal stage for said optical means on an underlying surface;
(c) a focussed radiant energy beam means auto-matically directing a beam at a selected first energy intensity and a selected second energy intensity in a path determined by and in focal registry with said optical means focussed upon said cells on said film support surface, said beam and said surface in relative automatic movement to expose all of said cells to said beam;
(d) discrimination means automatically identifying individual cells upon said cell support surface in accord with selected chemical, physical, or dynamic cell properties as elicited by said beam at first energy intensity and determined through said optical means; and (e) automatic control means connected to said radiant energy beam providing selected plural energy levels of said beam to said film support for selected irradiation, circumscription and isolation of selected cells on said film support surface as indicated by said discriminating means and isolation of selected encountered cells by a circumscribing application of a selected second energy intensity of said beam.

10. The apparatus of claim 9 wherein said support is a plate covered by a film on said plate beneath said cells and said film is bondable by said beam to said plate at selected energy intensity of said beam and isolating cells within a circumscribed area; and said film thus circumscribed removable from the said plate to achieve removal and destruction of unwanted cells from said plate.

11. The apparatus of claim 9 wherein automatic control means selectively adjusts the intensity of said radiant energy beam and selectively changes the energy intensity for irradiation, identification, isolation and selected destruction of unwanted cells.

12. The apparatus of claim 9 in which selected drive means is coordinated with said support means for selected automatic and manual coordination with said detection means and said radiant energy beam to sort out and save selected cells and destroy selected other of said cells and the organic residue from said destruction being isolated from said saved cells.

13. The apparatus of claim 9 wherein the discrimin-ation means to identify wanted cells from unwanted cells on the support comprises a photomultiplier tube responding to selected physical, chemical and dynamic properties of some of said cells and not others to identify selected cells by responding to cell reaction to said beam attenuated to signal the selection,

14. The apparatus of claim 9 wherein the detection means is photomultiplier tube in coordination with said optical path and beam focal path and sensitive to selected levels of luminescence from said cells on said stage as excited by said focussed radiant energy beam and providing a tracking path to selected of said cells under selected subsequent selected intensity levels.

15. The apparatus of claim 13 wherein a memory records said location of said cells and controls selected return to said location at a selected circumscribing and isolating energy entensity for circumscription of selected of said cells and for isolation therefrom of other and un-selected of said cells.

16. The apparatus of claim 9 wherein the identi-fication means is photomultiplier tube which coordinates with the position of said cells on said support surface at selected luminescence in a first irradiation at a first energy intensity trace of said beam and thereupon returning to said selected cells at higher beam intensity excising certain of said selected cells and leaving circumscribed cells segregated in situ.

17. The apparatus of claim 9 wherein programmed computer means are provided responsive to sensing by a photomultiplier tube during scan of said cells in situ on said film on said support surface at selected energy intensities, said computer means controlling said beam and its selected attenuation firstly to irradiate said cells in a prearranged path on said film support surface and then in selected isolation and removal of one group of said cells as selected in situ on said film.