METHOD AND APPARATUS FOR STUDYING THE REACTION PATTERN OF CELLS/CELL AGGREGATES DURING PERFUSION BY A TEST MEDIUM

Abstract: This invention relates to a method of studying the reaction pattern of cells/cell aggregates, for instance cancer cells, by microscopy and with a high time resolution during perfusion with at least one test medium in liquid state, for instance containing cyto-toxic drugs. According to the invention the cells/cell aggregates are introduced into a small space (17) in a perfusion chamber and are perfused by means of a control medium, the normal motility and reaction pattern of the cells/cell aggregates in the control medium being studied and recorded. Thereafter the cells/cell aggregates are perfused by means of the test medium, which exchanges the control medium with a distinct time resolution, the motility and reaction pattern of the cells/cell aggregates in the test medium being studied and recorded. Hereby important information about cell shape, cell size, cell motility, cell death, cell fluorescence etc. is obtained. The invention also relates to an apparatus which is usable for carrying out the method. This apparatus comprises a larger space (3) which serves as a reservoir for the perfusing medium and a smaller space (17) for the cells/cell aggregates through which space the medium is intended to be perfused. The apparatus further comprises a microscope by means of which the motility and reaction pattern of the cells/cell aggregates can be studied during the perfusion, and a sucking means (32) by which a medium can be sucked out of the space (17) and another medium can be sucked into the space (17).

Description: METHOD AND APPARATUS FOR STUDYING THE REACTION PATTERN OF CELLS/CELL AGGREGATES DURING PERFUSION BY A TEST MEDIUM The present invention relates to a method of studying the reaction pattern of cells/cell aggregates, for instance cancer cells, by micropscopy and with a high time resolution, during current perfusion (medium flowing by the cells) with at least one test medium in liquid state, for instance containing cyto- toxic drugs. The invention also relates to an apparatus usable to carry out the method. To date, cancer patients are usually treated with cyto- toxic drugs according to specific programs of treatment. These programs are according to previous knowledge regarding the therapeutical spectrum, of that drug or combination of drugs. Guided by the cancer proliferation the dosage and the type of drugs can be modified, however, usually with a considerable delay in time for the patient. The present invention intends to provide a new efficient method of analysis of the kind mentioned by way of introduction and is characterized by the combination of the following moments. - The cells/cell aggregates are introduced into a small space in the perfusion chamber, which is separated from a re¬ maining, larger space in the chamber by a slide, which space functions as a reservoir for the perfusion medium; - The cells/cell aggregates are perfused with a control medium, introduced into the larger space by a sucking means at the same time as the normal motility- and reaction pattern of the cells/cell aggregates in the control medium is studied by microsopy and recorded by means of photometry, photography, and/or video; - The sucking means is stopped and at the same time the larger space in the chamber is drained from the control medium, after which the test medium is added to the space; - The sucking means is started, the control medium in the small space being sucked out of said space, and a straight interface of test medium will simultaneously be suched into the small space without mixing with the control medium, for reac- tion with the cells/cell aggregates; - During the subsequent perfusion of the cells/cell aggregates it is possible to rapidly and with a high time resolution study the motility- and reaction pattern of the cells/cell aggregates in the test medium by means of microscopy and record by means of photometry/photography/video; - The photometry, photography, and/or video recordings are studied by a computerized image processor, whereby informa¬ tion regarding cell shape, cell size, cell motility, cell death, cell fluorescence, etc. is obtained. It is hereby made possible for hospital personnel to rapidly determine the response of individual cancer cells to different cyto-toxic drugs outside the body of the patient. Furthermore, it is possible to find the optimum combination of cyto-toxic drugs, already at the time for the onset of the cancer disease for the treatment of the patient. The rapid finding of the optimum treatment can be of critical importance to the survival of the patient. To safely determine an abnormal motility- and reaction pattern of the cells/cell aggregates during perfusion with a test medium, this perfusion should follow on a corresponding perfusion with a control medium. Thus the method allows the study of the said reaction pattern of the cells/cell aggregates by means of a high time resolution as the straight interface of test medium washes away the control medium. A preferred embodiment of the invention, usable to carry out the method, is herein more closely described with reference to the accompanying drawing, in which: Fiσ. 1 shows a plan view of the apparatus according to the invention, Fig. 2 shows a longitudinal section along the line II-II of the main part of the apparatus, a perfusion chamber together with a microscope objective, and Fi . 3 shows a preferred flow damping means. Referring to the drawing is shown there the new appara¬ tus, which comprises a perfusion chamber as a main part. This one comprises a frame 2, essentially of rectangular shape and preferably made of plastic material, and is designed to include an open space 3, essentially of rectangular shape. The frame 2 comprises two long-sides 4, 5 which according to this embodi- ment have essentially a square cross section and a width which is less than the width of the two short sides 6, 7 of the fram 2, which ones according to this embodiment have essentially square cross section and are essentially of standard dimension for microscope slides (approximately 25 x 75 mm) . According to the invention it is of advantage if the width of the frame parts 6, 7 is relatively large, to provide medium inlets to an medium outlets from the perfusion chamber formed by the frame via inlet channels 8, 9 and an outlet channel 10, which pass through the frame parts 6, 7 from their top surface to their bottom surface. The orifice of the channels 8, 9 is located at the border portion between the bottom portion of the part 7 an the space 3, whereas the orifice of the channel 10 is located at the border portion between the space 3 and the bottom por- tion of the part 6. The frame 2 is intended to be placed to a transparent bottom slide 11, of glass or plastic material, the latter bein placed on a microscope stage 12. The chamber frame 2 is prefer¬ ably glued to the bottom slide 11. The microscope stage 12 has an opening 13, in which a microscope objective 14 is intended to be positioned under the bottom slide 11 (inverted micro¬ scope) or above the chamber 1 (conventional mircoscope) . The front part of the frame 2, with reference to the flow direc¬ tion, is intended to be located above the opening 13. This means that the front half of the space 3 will be in line with the microscope objective 14. A transparent thin cover 15 of glass or plastic, which can be made in two pieces having a circular cut out for the objective 14 if the microscope ob¬ jective has to reach down into the chamber 1 (meant for con- ventional microscope), is placed on top of the frame 2. Due to that fact evaporation from the chamber is avoided and the chamber 1 is closed to contain a medium. Just above the opening of the channel 10 in frame part 6, in the space 3, a thin transparent slide 16 is positioned, which is intended to extend backwards, in relation to the intended flow direction, at a short distance from, and essen¬ tially in parallel with, the bottom surface plane of the frame 2. An extremely small space 17 is formed herein in which cells/cell aggregates, for example cancer cells, can be intro¬ duced. This space 17 have a volume of about 60 μl according to the preferred embodiment. It is of course possible to increase this volume by increasing the distance between the bottom sur- face of the frame 2 and the slide 16. The larger space 3, out¬ side the small space 17 of the chamber 1 functions as a medium reservoir, and also functions to stabilize the temperature in the chamber 1 as such and, in particular, in the small space 17. The medium in the chamber 1 is intended to absorb a major part of the heat generated by the microscope light. The appara¬ tus includes a thermistor probe 18 that reads the temperature in the chamber 1. In the event of long perfusion experiments, with an insufficient chamber medium reservoir, additional medium can be supplied to the chamber 1 via the inlet channels 8, 9 connected to external reservoirs 19, 20. Thus the external reservoir 19 is connected to the chamber 1 by a valve 21 and the inlet channel 9 whereas the external reservoir 20 is con¬ nected to the chamber 1 by another valve 22 and the inlet channel 8. A syringe is connected to each of the two valves _21, 22 the function of which syringe is to more easily fill the system with medium and, after experimental use, to clean the chamber and tubing. The tubing 25, 26 between the reservoirs 19, 20 and the chamber 1 can be equipped with a heat-exchange means 27, as indicated in Fig. 1, by which the medium to the chamber can be cooled or heated to an appropriate temperature. The heat-exchange means 27 may either comprise a separate heat-accumulator, being placed on the microscope stage 12, and through which the tubing 25, 26 is threaded, or by the micro¬ scope stage 12 as such, being in contact with the tubing 25, 26, and which functions as heating means. The tubing 25, 26 can be threaded into a tube of wider diameter for insulation. Introduced into space 17 is a needle 28, which is con¬ nected to a syringe 29, by which means cells/cell aggregates can be injected into the space 17. The chamber 1, which functions as a reservoir to the medium, can be emptied by means of one or more needles and/or drain channels 30, which are inserted into/debouching into the chamber 1, and which are connected to a vacuum source, for instance a syringe 31. Downstreams from the chamber 1, with reference to the flow direction of the medium, is a sucking means 32 positioned. By this sucking means the medium in the chamber 1 can be made to perfuse the cells/cell aggregates in the space 17 and further to the outlet channel 10, the tubing 33, and the suck¬ ing means 32 to a waste 34. Between the sucking means 32 and the chamber 1 a flow damping means 35 can be connected, the function of which is to eliminate vibrations from the sucking means 32. Such vibrations could dislogde the cells in the space 17 from the bottom of the chamber 1 because the cells are attached to the glass/plastic slide 11 by simple adhesion. The cells could also be allowed to be firmly attached by growing to the bottom slide 11 of the chamber or to a separate glass slide, which is introduced into the space 17, or to be held by means of a micropipette, which has been introduced into the space 17. A preferred embodiment of the flow damping means is shown in Fig. 3. The flow damper includes a cylinder-like container 36, which is covered by a top plug 37, and which is intended to hold both the liquid medium 38 and air 39. The air is intended to function as a damping medium. The connection between the sucking means 32, via the outlet tube 40, and the liquid 38 in the container 36 is made by means of a vertically adjustable tube 41. Because the tube is adjustable it is possible to vary the air to liquid volume ratio in the container 36 and hence the damping result. The apparatus may also include a syringe with valve 42, which is placed between the flow damper 35 and the chamber 1. Hereby, the space 17 and the reservoir 3 can be flushed by a medium which is taken out by the syringe 42. As previously described, the chamber 1 is equipped with a top cover 15. By simply removing the top cover 15 good access is provided to the slit-shaped opening between the cell space 17 and the remaining space 3 in the chamber 1. Thus, cells can be easily added and washed away between the experiments. Furthermore, micropipettes can be introduced into the cell space 17 for the manipulation of cells during current perfu- sion. This option opens another application for the new inven¬ tion. The apparatus can also include an air channel 43, which goes through the frame part 7, and is intended to connect the space 3 of the chamber 1 to the atmosphere. This air channel 43 makes it possible for the cover slide 15 to remain in position during the experimentation, which can be of advantage when using dangerous cyto-toxic drugs, harmful to personnel. The apparatus of the invention is used as follows: Cells, for example cancer cells, are introduced into the space 17, followed by a perfusion for an appropiate period of time, for instance 15 minutes, by means of a control medium which has been added to the chamber 1, for instance, from the first reservoir 19. The motility- and reaction pattern of the cells/cell aggregates is studied during the control perfusion. The cells are simultaneously recorded by photometry, photo¬ graphy, and/or video to be analyzed by a computerized image processor. Hereby, cell shape, cell size, cell motility, cell fluorescence, and/or cell changes of other kinds can be effi- ciently studied. The chamber 1 is then drained from medium by the syringe 31, after which the connection between the chamber 1 and the second reservoir 20 is opened, the latter holding a test medium for instance- containing cyto-toxic drugs in liquid state. The chamber 1 is hereby filled with the test medium. The medium surrounding the cells in the space 17 will not be affec¬ ted during the draining of the first medium from the chamber because the sucking menas 32 is not operating during the medium exchange. The time needed for the medium exchange procedure as such is usually 5 to 10 seconds. When the sucking means 32 is restarted, a distinct medium exchange will take place in the cell space 17 because of its small volume and because a close to straight interface of test medium moves towards the cells and pushes the control medium, remaining in the space 17, ahead. Thus will the cells be per- fused again, but this time with the test medium, that contains a cyto-toxic drug, and simultaneous changes to the cancer cells due to the exposure to the cyto-toxic drug can be studied by means of microsocpy and recorded by photometry, photography, and/or video. A high time resolution is hereby provided (se¬ conds) for the study of acute, rapid changes to cells in re¬ sponse to test compounds. It is of course possible to modify the apparatus within the scope of the claims. For instance, the chamber frame 2 could be made of stainless steel instead of plastic. Further¬ more, the chamber could be in one piece instead of letting the frame 2 be attached to a bottom slide 11, and a top cover 15. The frame 2 can also be made in two pieces with a detachable short end module which is pressed in position at the place for the short side 7 and which is equipped with the holes for the inlet channels 8, 9, thermistor probe 18, drain channel/drain channels 30, air channel 43 and other possible channels. This is in order to simplify the replacement of the chamber which can be made for single use and disposed after use. The frame 2 can also be provided with a mm-scale 44 along the longitudinal axis of the frame 2, above and along both sides of the cell space 17 to determine the location of the cells under study in relation to the opening between the cell space 17 and the e- dium reservoir 3. This distance affects the time of cell ex¬ posure to the test medium in relation to the time when the sucking means 32 is started after the medium exchange in the reservoir 3. For use in a conventional microscope, with the objective 14 coming from above instead of from below, according to Fig. 2, the chamber 1 can also be designed for use upside- down on the microscope stage 12. In this connection it is needed to modify the channels 8, 9, 10, and 43, to be access¬ ible in the frame 2, and the slide 15 must be attached to the frame 2 whereas the slide 11 is made detachable instead. The enclosed volume of the chamber 1 can be varied by means of a special insert, preferably made of stainless steel, to fit in the space 3. In the insert two holes are drilled to connect to the channels 8, 9 and eventually to the drain channel 30. In addition, the insert can function as a heat accumulator. The insert, including its channels, can also be designed to allow control and test media to be distributed over the entrance of the cell space 17. The apparatus, with or without insert, can also be made to allow control and test medium, respectively, to be added by an additional pump in excess to the flow that enters the cell space 17 by the sucking means 32. The excess medium escapes from the chamber l via the drain channel 30. By this procedure, test medium can flush towards the entrance of the cell space 17 without the need for the reservoir 3 of the chamber to be drained at medium exchange. Furthermore, the orifice of the channels 8, 9, 10, 43 and one or more drain channels 30, can be made conical to fit a conical nozzle for a corresponding tubing.

Claims: Claims 1. Method of studying the reaction pattern of cells/cell aggregates, for example cancer cells, by microscopy and with a high time resolution during current perfusion with at least on test medium in liquid state, for instance containing cyto-toxi drugs, c h a r a c t e r i z e d by the combination of following moments: - The cells/cell aggregates are introduced into a small space (17) in a perfusion chamber (1) , which is separated from a remaining, larger space (3) in the chamber (1) by a slide (16) , which space (3) functions as a reservoir for the perfu¬ sion medium; - The cells/cell aggregates are perfused with a control medium introduced into the larger space (3) by a sucking means (32) at the same time as the normal motility- and reaction pattern of the cells/cell aggregates in the control medium is studied by microscopy (14) and recorded by means of photometry, photography, and/or video; - The sucking means (32) is stopped and at the same time the larger space (3) in the chamber is drained from control medium, after which the test medium is added to the space (3) ; - The sucking means (32) is started, the control medium in the small space (17) being sucked out of said space, and a straight interface of test medium is simultaneously sucked into the small space (17) without mixing with the control"medium, for reaction with the cells/cell aggregates; - During the subsequent perfusion of the cells/cell aggregates it is possible to rapidly and with a high time resolution study the motility- and reaction pattern of the cells/cell aggregates in the test medium by means of microscopy and record by means of photometry/photograph /video; - The photometry, photography, and/or video recording are studied by a computerized image processor, whereby information regarding cell shape, cell size, cell motility, cell death, cell fluorescence, etc. is obtained. 2. Apparatus usuable for carrying out the method accord¬ ing to claim 1, to study the reaction pattern of cells/cell aggregates by microscopy and with a high time resolution, for instance the reaction pattern of cancer cells during current perfusion with different media in liquid state, for instance containing cyto-toxic drugs, c h a r a c t e r i z e d by the combination of following features: - A chamber (1) comprising a first larger space (3) , which serves as a reservoir to the medium, and a second smaller space (17) for the cells/cell aggregates and through which the medium is intended to be perfused; - A microscope stage (12) equipped with a slide (11) , being at least in part transparent and intended to function as the bottom slide (11) for the chamber (1) , the part of the chamber (1) holding the second space (17) being intended to be positioned above the transparent part of the slide (11) , and a microscope objective (14) being intended to be located at an opening (13) in the stage (12) under the slide (11), or to be located above the space (17) ; - A first sucking means (32) is located downstream in relation to the chamber (1) and with reference to the intended flow direction, the sucking means being in connection with the second space (17) , via an outlet channel (10) which channel is within the chamber (1) . 3. Apparatus according to claim 2, c h a r a c t e r ¬ i z e d in that the chamber (1) is connected to at least one but preferably two medium reservoirs (19, 20) via inlet chan- nels (8, 9) and valves (21, 22). 4. Apparatus according to claim 2 or claim 3, c h a r ¬ a c t e r i z e d in that the chamber (1) consists of a frame (2) , which is intended to be in contact with the slide (11) by its bottom surface and to be covered by a cover slide (15) on its top surface. 5. Apparatus according to claim 4, c h a r a c t e r ¬ i z e d in that the microscope objective (14) is intended to be positioned under the bottom slide (11) , or in an opening in the top slide (15) , approximately centred in relation to the space (17) , and via a mm-scale (44) in a determinable position in relation to the opening of the space (17) to the medium reservoir (3) . 6. Apparatus according to claim 4 or 5, the front half of the chamber (1) , seen in the intended flow direction of the medium, being intended to be in contact with that part of the slide (11) under/above which the microscope objective (14) is intended to be located, c h a r a c t e r i z e d in that th second cell space (17) is formed by a thin slide (16) which is arranged in the bottom portion of the chamber (1) in its front half essentially parallel with the bottom slide (11) of the chamber (1) and at a small distance from the same. 7. Apparatus according to claim 6, the frame (2) compris- ing four parts, two (4, 5) longitudinal parts in the intende flow direction of the medium and two (6, 7) parts, transverse to the intended flow direction of the medium, c h a r a c ¬ t e r i z e d in that the cells/cell aggregates are intended to be introduced into the second space (17) of the chamber (1) by means of a syringe (29) via a needle (28) going through the frame part (5) and extending into the space (17) . 8. Apparatus according to claim 7, c h a r a c t e r ¬ i z e d in that the first space in the chamber (1) is intended to be drained by means of a second sucking means (31) which is in connection with the space (3) via one or several needles and/or drain channels (30) going through the frame part (4) and/or the frame part (5, 6, 7) and extending into the space (3). 9. Apparatus according to claim 8, c h a r a c t e r - i z e d in that the one external reservoir (19) is intended to fill the chamber (1) with a first medium, for instance a control medium, that the other external reservoir (20) is in¬ tended to fill the chamber with a second medium, for instance a test medium, the means (30, 31) being intended to drain the space (3) before the second medium is added. 10. Apparatus according to anyone of the claims 2-9, c h a r a c t e r i z e d in that a flow damper (35) is located between the first sucking means (32) and the chamber (1). 11. Apparatus according to claim 10, c h a r a c t e r ¬ i z e d in that the flow damper (35) comprises a cylinder-like container (36) which is sealed by a plug (37) and is intended to hold both the medium (38) and air (39) , that an adjustable tube (41) goes through the plug (37) and connects the tube (40) to the sucking means (32) with the liquid medium (38) in the container (36) , and that the tube (41) is vertically adjustable so that the liquid to air volume ratio in the container (36) can be varied. 12. Apparatus according to claim 11, c h a r a c t e r ¬ i z e d in that the sucking means (31, 32) comprise a pump (32) and a syringe (31) with valve, respectively, that the frame (2) is made of plastic material or steel, and that the slides (11, 15, 16) are made of glass or plastic material.

Inventors: ENGSTROEM GUNNAR, ENGSTROEM, GUNNAR
Applicants: ENGSTROEM GUNNAR, ENGSTROEM, GUNNAR
Application year: 1990
Main classification: C12M3
IPC codes: C12M1, C12M3, G01N33, G01N35