JPH10276763A - Compact cell culture disk - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide the subject disk capable of simultaneously, mutually and independently culturing many biological samples within a wide range and simply observing cells by installing many microchambers hermetically sealable from the surroundings with a selectively divided semipermeable inner wall. SOLUTION: This compact cell culture disk 1 is obtained by arranging divided microchambers 17 including pits hermetically sealable from the surroundings with a semipermeable inner wall in the central region of the disk 1. A cell affinity plane 18 may be provided at the center of each microchamber 17. Each pit is capable of forming a closed microlaboratory in which the inner environment is determined by properties of the permeability of a covering thin membrane.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an array of living spaces of microscopic dimensions isolated from one another, to a method for producing such an array, and to a method for investigation and measurement using this array.

[0002]

BACKGROUND OF THE INVENTION The present invention described herein is directed to a flat disk suitable for culturing microscopic biological specimens in the form of single cell organisms, cell aggregates or multicellular microorganisms. The design and use of microchambers in the form of shallow pits. One such "compact cell culture disc" (CCCD) is co-culture, cloning, instrumental or macroscopic observation, and storage of hundreds to thousands of biological samples, optionally isolated and confined (in vitro or postmortem). ). The goal is to enable efficient, precise and reproducible study of microscopic and physicochemical parameters in a sample set as described above under uniform culture conditions. For this purpose, the samples (single cells, fertilized eggs, embryos, etc.) can each be individually implanted in one isolated microchamber of the sample. The microchamber is made of silicon, quartz, glass, plastic or a suitable composite thereof and is made up of small holes (typically 500 mm diameter) in a thin biochemically inert film securely attached to the bottom plate. Shallow (typically 10-1 depth) defined at ~ 5000 micrometers
(00 micrometers). The pits can be covered with a thin plastic film formed from a semi-permeable coating (FIG. 1). In this way CCC
Optimal and uniform breathing gas, nutrients, antibiotics for D
Control of reagents and the like can be guaranteed. In this way, each pit can provide a closed, sterile microchamber whose internal environment is determined by the permeability properties of the coating membrane. The macromolecular media component can be injected through the coating before or after the pit is closed. The above-described arrangements provide excellent conditions for individual culture and microscopic or instrumental analysis of microscopic biological samples. In addition, exposure to physical, chemical or biological agents can be controlled for laboratory or other uses. When coated with a thin film that is impermeable to viruses and bacteria, the chamber forms a closed biological system and functions as a virtually sterile micro-laboratory.
After culturing and after the experiment, the investigated substances can be fixed or frozen for further analysis or storage without dislodging from the CCCD.

[0003] Biological microsamples in various biological specialties (cell and molecular biology, microbiology, genetics, immunology, oncology, radiation biology, pharmacology, embryology, etc.) There is a need for individual culture, growth characterization or other cell biological parameters, cell cloning and clone selection and documentation, recording and archiving (in vitro or post mortem). For this reason, the description herein, in addition to the description of the invention itself, demonstrates the adaptation of CCCD to microscopy and physicochemical measurements.

[0004] The techniques described above have found application in the fields of basic biomedical and biotechnological research and their continued development and practical application, for example in clinical diagnostics and therapeutic planning, toxicology and fetal hygiene, and in the field of industrial genetic engineering. Indispensable for efficient use of the present invention. CCCD
Provides particularly favorable conditions for the production of daughter cell populations from single cells (cell cloning). Under uniform culture conditions, it is possible to produce a large number of cell clones and to provide, for example, 1000 to 10000 cells in a certain size. This situation has significant benefits, especially in genetic engineering research and tumor diagnosis.

[0005] In tumor diagnosis, for example, a tumor biopsy, which typically contains hundreds of thousands to millions of tumors and stromal cells, is adequately represented by the growth of thousands of cell clones simultaneously in the same CCCD. Can be done.
To this end, dispersed single cells obtained from the tumor can be automatically implanted in individual micropits. Optionally, one can re-sort to random implantation with a highly diluted cell suspension. This is usually 1
This is a technique that leads to successful implantation of 1 or 0 cells per pit.

The existing microchamber can be used as a cell biologically aseptic laboratory because the coating film of the pit can be impermeable to bacteria, viruses and other biological contaminants. A problem arises when nutrients and other substances are exchanged very limitedly through this cover film. However, the proposed exchange structure can be eliminated or suppressed by the proposed individual culture structure and minute cell mass.

[0007] One of the objects of the present invention is to cultivate a large number of biological samples utilizing a certain sequence at the same time and widely independently of each other.
At the same time, make the array observable in the simplest way. In the case of a single cloned cell population, a small micro-laboratory must be constructed which allows favorable conditions, especially for culture and analysis. This allows
For example, when the genes are homogeneous, an advantage must be provided that allows for reliable characterization of representative selection of clonal cells from tumor samples containing hundreds of thousands or millions of cells. The problem according to the invention has to be solved, for example in clinical oncology, by establishing individual locations for at least about 1000 clones, individual and isolated from each other. This creates the conditions for the desired representative of the selection described above. This sequence must of course be available in a single microlab from small cell populations to single cells for culture breeding, research and storage.

Another problem is that this sequence can be supplied to individual cells in the simplest way, and all effects on the uniformity of growth conditions during culture and during observation of growth and cell properties. The removal of unwanted external influences within the microchamber or individual "pits".

[0009] Yet another object is to construct a suitable assay for recording or evaluating the sequences constructed according to the present invention or the cells or biological cultures arranged therein during the growth phase. is there.

[0010]

The object set according to the invention is solved in particular by using the arrangement according to claim 1.

According to the present invention, cells having a plurality of microchambers or "pits", which are isolated from each other, and which are arranged in a honeycomb shape with the precision of microscopic observation on, for example, a disk-shaped flat pedestal, are cultured. An arrangement for doing so is proposed. The microchamber is covered by a semi-permeable inner wall or thin film. That is, it is sealed against surrounding biological contaminants. Chamber structures are typically so-called sandwich structures of parallel plane, thin plates and / or thin films in combinations selected for various application purposes. At that time, the plate or the thin film arranged at the center of the sandwich structure is partially perforated to form a microchamber in a mesh shape or a honeycomb shape. At the same time, a pedestal, which can be part of a "sandwich structure", is a disk formed, for example, by a thin silicon structure, a quartz structure, a glass or plastic structure. The area diameter of the cradle can typically be 5 to 20 cm, depending on the number and size of the microchambers disposed thereon. By selecting a circular cradle, an embodiment of the array that can be best compared to a compact disc used in the audio or video field, which can be scanned, for example, using the analysis of a microchamber by a microscope or a spectroscope This leads to the possibility of a variant.

For example, a micro-chamber arranged in a honeycomb shape typically has a diameter of about 0.5 to 5 mm,
However, it is not necessarily circular and can be finished.
Between the honeycomb structure, which defines the geometry of the chamber, which also determines the distribution, shape and size of the chambers, and the disc-shaped cradle, if preferred, for example, an agarose layer (Agaro layer)
A thin transparent film, such as a schschicht, or a thin layer of transparent gel can be placed without cell affinity. Of course, other suitable substances that are widely inert to the cell or its culture or have other properties to form the layer can also be used.

Suitable for culturing cells, such as a centrally formed “Palladiuminsel”, which is mounted on the bottom or pedestal of each microchamber of the honeycomb structure or on the above-mentioned thin film or gel layer. A typically concentrically formed cell-friendly plane, consisting of a thin cell-friendly metal or molecular layer, or other material forming the same function plane, can be provided. The honeycomb structure itself or the cabin wall can be manufactured from dimensionally stable materials such as, for example, plastics, metals, ceramics or composites. Finally, the semi-permeable membrane preferably consists of a material that is at least almost transparent to light, such as, for example, Teflon, polycarbonate or polystyrene.

[0014] In another preferred embodiment of the sequence according to the present invention, a variant and a method for producing a sequence for culturing cells are described in claims 2 to 20.

The individual method means for producing the sequences according to the invention will be explained in more detail in connection with the figures described below. A variant of the preferred embodiment of the method according to the invention is characterized in dependent claims 14 to 18.

The present invention or sequence is also described in the "Compact Cell Culture Disk"
Or "CCCD"), in small or large quantities, ie, from single cells to typically thousands of cells under physically, chemically or biologically optimized conditions. Enables microscopic, molecular biological, biochemical and physical chemical studies of individual cells or cells or multicellular structures. With the biological material implanted in the proposed cell culture array, a micro-laboratory is created that allows the control, observation and analysis of individual cultures or their formation. The environment can be controlled individually or globally. Both the entire sequence and the individual compartments or microlaboratories, under sterile or non-sterile conditions, for short or long periods, and also for permanent storage, for transport or for observation purposes, for example under a microscope or by spectroscopic methods, For example, it can be held by using a light guide, a photomultiplier or a photodiode.

[0017] Finally proposed are information on the cells or cell culture clones and other biological samples mentioned, which have been implanted and stored in a sequence defined according to the invention, measured or recorded, evaluated and possibly also according to the invention. Is a way for memorizing. These are preferably automated electronic data processing (EDV) assisted methods, which can increase implantation and analysis capacity and speed in the context of manual and visual techniques.

[0018]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The embodiments of the present invention shown in the attached drawings will be described below.

FIG. 1 (a) is a plan view showing a "Compact Cell Culture Disk" and an example of "Kompaktzellkulturscheibe bzw.-platte" in German is shown in FIG. 1 (b) and FIG. c) with microchambers in variants of the two embodiments depicted in c).
The structure of this disc 1 will be described in more detail below with reference to FIG. Arranged in the central area of the disc 1 are microchambers or microlabs 17 or 18 defined according to the invention, each of which has a so-called pit, which can be closed off. At that time, a cultured cell or cell culture can be arranged at the center of each microchamber or each pit. Finally, for reasons of manufacturing technology, the disc 1 has a groove or notch 19 at the periphery, the significance of which is likewise evident from the following description.

The arrangement of the microchambers 17 shown in FIG. 1A is only an example, and, as will be apparent from FIGS. 10A and 10B described below, the It is also possible to arrange the chamber 11 on the disc 1 in any other way.

An excerpt from the disk 1 of FIG. 1 (a) shows a microchamber 17 in each of FIGS. 1 (b) and 1 (c). The microchamber of FIG. 1 (c), which is not present in the microchamber of FIG. 1 (b), is provided with a cell affinity plane 18 at the center. Various types of such planes, locations and finishes and various locations or other cell-affinity structures can be used as well, as freely floating particles.

In connection with FIG. 2 and FIGS. 3 and 4,
In cross section, variants of various embodiments of the possible arrangement according to the invention are shown. FIG. 2 schematically shows the components or individual elements specified in the structure of the above-mentioned arrangement. Considered for the structure of the arrangement are, as shown in FIG. 2, a metal, ceramic or plastic film and a semi-permeable inner wall 5 formed, for example, in a honeycomb or perforated manner. This can consist, for example, of Teflon, polystyrene or other suitable, preferably light-transparent, material. To form a true micro-chamber or micro-laboratory, a grid-like or honeycomb-like matrix 6 made of a dimensionally stable material such as, for example, plastic, metal, ceramic or composite material or reinforced Duromeren is used. used. However, the matrix can also be manufactured from a silicon disk which has been treated using oxidation or etching, as indicated for example by the reference numeral 8 in FIG. In this case, the oxidized or etched silicon matrix is preferably arranged in a transparent quartz layer.

As cradle for the matrix 6, it is possible to use, for example, a silicon disk 7 provided with a cell-friendly plane 18 on which cells to be cultured can be arranged. Furthermore, this cradle can be part of a silicon disk that is processed using oxidation and etching, for example a disk already having the reference number 8 in FIG. 2 described above. However, this cradle 7 can also be manufactured from quartz or glass instead of silicon, or from other suitable materials. Preferably, the silicon cradle is coated with a thin agarose polymer layer, on which the cell-affinity spots 18, for example made of palladium, are arranged, with the agarose layer resting thereon. Unlike palladium, agarose has no cell affinity. The aforementioned palladium spots are particularly suitable for culturing and cloning individual cells. The so-called palladium insel technology will not be described in further detail here. This is because this technique is very well known in the relevant state of the art for optimally culturing individual cells. Here I will point out only the reference: U. Ama
ldi, B. Larsson: Proceedings of the First International Hadron Therapy Symposium, "Hadron Therapy in Oncology". Como, Italy, October 18-21, 1993 Excelta Medica, International Conference Series 1077, 1994, Elsevier, p. 735 et seq. (U. Amaldi, B. Larsson, Hadron
therapy in Oncology, Proceedings of the first inte
rnational symposium on Hadrontherapy, Como, Italy,
18.-21. October 1993, Excerpta Medica, Internat
ional Congress Series 1077, 1994, Elsevier, Seiten
735 ff.).

Of course, other similar methods can be used for this purpose. Depending on the respective arrangement selected, it is also possible to magnetically hold the sandwich structure for which the use of the magnetic disk 9 is considered. The magnetic disk can be formed flat as indicated by reference numeral 9 and can also penetrate in a honeycomb or hole shape as indicated by reference numeral 10. The latter magnetic disk is necessary, for example, when optical analysis is performed through the magnetic disk.

In order to ensure the storage, handling and transport of the sandwich-shaped array or analysis structure according to the invention, provision is made for a container 11 which can be, for example, a special steel box provided with an observation window on at least one side. Is preferred.

Referring now to FIG. 3, a container 11 including an array 12 is shown in cross-section. For clarity, the array 12 has been exploded and is also shown in FIG. This clearly shows the components used in the individual FIG. At this time, the structure or array 12 corresponds to a cross section along the line AA in FIG.

The structure or arrangement 12 consists primarily of a honeycomb-shaped or perforated silicon disk, which can be combined with a quartz foil or quartz plate 8. The hole-shaped opening of the silicon disk 8 can be obtained from the silicon disk by oxidation or etching. The quartz surface is preferably coated with a layer of agarose polymer whose material has no cell affinity. The structure according to FIG. 3 further comprises a film 5 covering the honeycomb and a newly formed nickel film 4 in the form of a honeycomb. At this time, the honeycomb structure is the same as the structure of the silicon disk 8. In order to retain the structure or arrangement according to the invention, a magnetic disk 10 is arranged in the quartz layer opposite to the silicon honeycomb, which is again combined with the silicon honeycomb provided with the corresponding opening. Provided. The arrangement of the metal foil and the magnetic disk maintains this arrangement as a result of the magnetic force. Finally, for secure storage, this arrangement is inserted into a container 11, for example made of special steel, and stored therein. At the same time, the steel box can be provided with a window-like opening on one or both sides, which is again congruent with the silicon honeycomb structure. This window-shaped opening is used for analyzing or observing the array or structure or the cells or cell culture arranged therein by means of a suitable analytical instrument 21 or 21a. As an example of a cell-friendly structure, the individual chambers have a center of palladium insell (palladium compartment) 18 on the quartz surface, as shown particularly in the structure brought together.

Individual cells 2 on this palladium insel
3 can be attached, on which cell culture is started. The individual compartments are mixed with various liquids such as serum, nutrients, agents and the like. Since this structure is covered or sealed using the semi-permeable inner wall 5, it is also possible to insert other substances into the chamber during the culture process. The semi-permeable inner wall may be made of, for example, Teflon, polystyrene or other suitable transparent material.

FIG. 4 shows a new assembly together and another explosively developed arrangement according to the invention,
Shown schematically in cross-section and partial views. Furthermore, this partial view corresponds, for example, to the section AA in FIG. 1 (c), wherein the cells administered to this structure are not shown in FIG. In the arrangement or structure according to FIG. 4, a matrix 6 made of, for example, polycarbonate is mounted on a silicon disk 7. Further, the matrix is covered with the film 5, and the whole arrangement is held by the magnetic force between the metal foil and the magnetic disk. In order to store this arrangement, a new container 11 is prepared. At this time, in the example shown in FIG.
The analysis is considered through the cap of In this example, it is preferable that a silicon disk be newly provided using an agarose device. Further, a palladium insell 18 is provided on the agarose layer for inserting cells corresponding thereto.

Of course, in both the structure 12 according to FIG. 3 and the structure 13 according to FIG. 4, it is possible to select significantly different dimensions when forming the individual microchambers. The following dimensions have proven to be appropriate: Diameter of silicon disk: about 10 to 20 cm Thickness of silicon disk: 0.3 to 1 mm Thickness of agarose layer: <10 μ Inner diameter of microchamber 11 preferably having a circular shape: about 0.5 to 5 mm, preferably About 0.8 to 2 mm Thickness of honeycomb structure between individual small chambers: about 0.2 mm Height of inner wall of honeycomb structure: about 0.1 to 2 mm Diameter of palladium insel: about 0.3 mm For example, polymer coating made of Teflon Thickness of layer or semi-permeable membrane: <10 μ FIG.
1 schematically shows a partial view in a cross-sectional view of a variant of another possible embodiment of the arrangement 14 according to the invention provided for a measuring method for analyzing through one bottom. This structure comprises a new silicon quartz disk 8, a metal foil 4 and a membrane 5 arranged on the bottom of the container 11. This structure is covered by a magnetic disk 9, which at the same time forms the cap of a container or a special steel box 11.

The compact cell culture arrangement or structure 12 to 14 described according to the present invention, including the chamber structure described in detail in FIGS. 3-5, is particularly suitable for examining tumors containing a large number of different cells. First, the cell deposits of the tumor sample (biopsy) are dispersed and divided into individual cells. Indeed, it has been shown that a relatively large number of such individual cells need to be examined. Since samples such as those described above generally contain from about 100,000 to 1 million individual cells, it is desirable to examine at least several thousand individual samples to obtain a significant image of a tumor biopsy. The separation and dispersion of cell biopsies as described above is very well known.

The individual cells obtained by the above-described dispersion are now administered in individual, unsealed pits 17. Finally, the honeycomb structure has a semi-permeable inner wall 25.
To the outside using palladium insel 1
8 sealed. After filling and sealing the chamber 17,
Generally, standardized, small molecule growth components or nutrients are administered via a coating. An advantage of the arrangement or structure described according to the invention is that it has a large number of compact, biologically sterilized micro-laboratory, which opens up the following possibilities. 1. Transplantation (from thousands of samples) of any number of individual cultured cells in individually self-sealed chambers that can be subsequently observed and analyzed visually and instrumentally. 2. 2. Culture under optimal optical conditions using an upright or inverted, conventional or confocal laser scanning microscope and morphological or spectroscopic investigation of the environmental medium of the culture. Possibility of adding standardized small molecule media components such as buffers, nutrients, antibiotics. 4. 4. Removal of waste products delivered by organisms and growing cells through semi-permeable inner walls 5. Management of individual chambers with breathing gas or other gas through the semi-permeable inner wall again. 6. Continuous or primary addition of small molecule agents such as drugs or toxic environmental factors 7. Control of the individual biochemical and, if important, microbiological environment for each culture at the time of implantation or at any time using microinjection through the coating membrane. 8. Common or different effects of non-chemical test modalities such as ionizing or non-ionizing radiation at any time utilizing external exposure. 9. Common or different temperature control Sterilization conditions for storage, transport, handling, analysis and culturing during observation in a non-sterile environment Schematically in Figures 3-5, how cells are observed or analyzed during investigation in individual compartments. Microscope or other optics 21 to illustrate
Or 21a is shown. In this case, for example, healthy cells form a so-called "monolayer" that can be easily observed with a normal microscope, whereas so-called transformed cells ("tumor cells") can form a "multilayer" structure There is. This can be better observed or analyzed using a confocal microscope.

The formation of the compact cell culture plate defined according to the present invention is based on the structural principle of a new type of cell culture microchamber and the arrangement of said microchambers. This limits the isolation, cultivation, characterization and documentation of individual live cells, manual or automated, set at scale
Cell populations or structural systems of cells, such as small multicellular organisms or tumor or tissue ex vivo cultures are also possible.

The compact cell culture plate described according to the invention, which is constructed in a sandwich on a simple disc-shaped element, necessarily entails very good application and handling advantages. One is that the compact cell culture plate is relatively small and compact, and the other is that a lot of information is stored on the compact cell culture plate, and this information is stored in the simplest manner with the best accuracy. Can be analyzed or called. As described above, the cell culture plate or disk as described above can be inserted into an analytical device or a processing device in a manner analogous to a compact disk, and precisely defines individual cell chambers using precisely defined coordinates. Can be analyzed or influenced. In this way, a large number of microlabs can be scanned relatively quickly, arbitrarily or systematically, for example stepwise, radially and with azimuthal disk movement. There are a number of applications for the compact cell culture plates claimed in accordance with the present invention, including those applied in molecular or cytogenetics, microbiology, tumor biology, toxicology, pharmacology and radiation biology. I have. "Compact cell culture discs" (abbreviation: CCCD) are suitable for the application of automated routine work in biotechnology, clinical medicine or environmental technology, for example in the field of education. Especially with greatly increased capacity, accuracy,
Whether reproducibility and sterilization are manual or computer controlled,
It allows the implementation of so-called "cell cloning", ie isolation, culture, characterization and documentation of individual clones.

FIGS. 6 and 7 schematically show two possible measurement or analysis methods. Based on this, cells to be cultured or cultured cells or cell culture can be observed or analyzed. FIG. 6 shows, in a schematic cross section, an arrangement 1 according to the invention with a microchamber or laboratory 17 which can be used for measuring upwards. Observation or analysis of the individual cell spots 23 is thus carried out in their original position during the culturing process, for example using the optical instrument 21.
At that time, other analytical instruments can be used. Since this measuring method can be repeated arbitrarily, it can be preferably applied during the culture process. In doing so, in order to scan all the compartments 17 considered in a particular selection or arrangement,
Whole compact cell culture plate or disc 1
Is scanned.

On the other hand, the entire coating of the original structure was removed for precision measurement or for the removal of the suspension of substances, which were sealed in FIG. 7, and the cell culture or the cultured biological sample was killed and fixed, possibly frozen. Dry or incinerated substance 25
Occurs in the form of Measurement or analysis of dead material
This is done using photon or neutron radiation that happens to occur in the direction of the arrow suggested by the arrow. The great advantage of this measurement method is that, on the one hand, it conveys the most accurate results and, on the other hand, the radiation impinges almost parallel to the substance to be examined, so that the underlying receiver, for example a silicon disk, This is to be done without any scattering in the table. Measurements utilizing other particle radiation or X-ray photons or thermal neutrons are very well known from the prior art.

Unlike the microchamber structures or arrangements 12 to 14 which are held in a sandwich by the use of magnetic force, which are partially shown in FIGS. It is also possible to manufacture using adhesion. This is necessary, in particular, when the resulting magnetic field is deemed to interfere with the cultivation of the cells, as shown diagrammatically in cross section in FIG. That is, in some cases, a sandwich structure can be disadvantageous if it is held using a magnetic field, and thus, in some cases, a compact cell culture plate or array or structure according to the present invention is shown schematically in FIG. To
That is, it is necessary to manufacture it without any magnetic field. The compact cell culture plate according to the present invention as described above can be manufactured preferably according to the processing steps described below. 1. Honeycomb-like structure or matrix 6 from a relatively stiff material such as polycarbonate, silicon, ceramics or Teflon-coated nickel with preferably circular holes provided to form the microchamber 17 Procurement. At this time, the highest possible package density is preferably selected so that as many microchambers 17 as possible can be formed in the smallest space. 2. The aforementioned lattice-like or honeycomb-like structure or matrix is adhered onto a thin polymer film 5 (membrane) made of, for example, Teflon or polystyrene. At that time, for example, a silicon derivative or UV is used as an adhesive.
Curable polymers can be used. Polymer thin film
The above-mentioned, optionally obtained, preferably individual, compartments 17
Is formed. 3. Individual pits 1 with nutrient solution, serum, active substance, etc.
Filling 7 4. Administration of individual cells 13 into the chamber 11 opened up On a silicon wafer or glass plate having a diameter of, for example, about 10 cm, a thin polymer layer, for example made of agarose, is first provided. A palladium insell or other structure can be applied or dispensed onto the silicon wafer or agarose layer in harmony with the honeycomb structure. 6. The silicon wafer produced in this way is mounted on the above-mentioned honeycomb structure, wherein preferably both the honeycomb structure and the silicon wafer are marked or notched.
9 (FIG. 1 (c)). Thereby, the silicon wafer is jointly mounted on the honeycomb structure. The bonding of the silicon wafer having the honeycomb structure is performed using, for example, silicon grease. 7. Finally, the manufactured array or compact cell culture plate according to the invention is rotated 180 ° to the position described in FIG.

As an alternative to this, instead of bonding, one may use a plastic film with a honeycomb-like structure and the other a honeycomb-like structure with a silicon disk, using the magnets described above. For this purpose, in the manufacture of the honeycomb structure described in FIG. 2 which is still open on one side, a polymer film forming a semi-permeable wall is applied over a grid-like nickel film or a special steel film, which It also has small holes corresponding to the holes.
Again, each metal foil and polycarbonate-
The side cutouts 19 in the honeycomb structure ensure that the individual cells 17 are visible through the nickel film or metal foil, for example for analytical purposes. After the mounting of the silicon disk 7, a magnet plate 9 is placed on the back of the silicon disk, so that the array or compact cell culture-sandwich structure thus formed is held using magnetic force. Finally, the newly formed compact cell culture plate is rotated 180 ° so that the individual compartments 17 are visualized for analysis or processing purposes through pores in the membrane. In addition to the options or the magnet film, the honeycomb-like structure can be made of, for example, nickel or other magnetic material instead of a polymer.

FIG. 10A shows another example of a so-called "compact cell culture disk". In this case, the entire plate is provided with a honeycomb-shaped structure or individual microchambers, except for a relatively narrow peripheral portion. Based on this method, for example, in the case of a disk having a diameter of 10 cm and a microchamber having a diameter of 0.8 mm, it is possible to arrange 8000 or more microchambers.

FIG. 10B is further suitable for the embodiment of the "compact cell culture disc" according to the invention, in particular for the storage, cultivation and analysis of cells or cell cultures or other biological samples of different experimental series. For example,
It is also possible to administer cells of the first sample in the region of the structure 33, while administering cells of the second sample into individual compartments or pits of the structure or region 35, and so on. It is also possible to administer reference cells to each intermediate region 34 or 44 between the main regions, and also to culture and observe cells that require only a relatively small number for culture.

Finally, FIG. 11 shows the application of the arrangement described according to the invention particularly suitable for radiological research and radiology.
For example, in a model structure 31 filled with water, FIG.
As shown in the figure, a compact cell culture disc 1 manufactured according to the present invention is inserted. Here it is possible to examine the biological effect with a coordinate function of the samples on different sample systems or different compact cell culture discs. This is done by exposing the model structure 31 containing the compact cell culture disc to electromagnetic fields, X-rays, photons, neutrons or any other radiation 30. Depending on where the individual microchambers 17 are respectively located in the coordinate system of the model structure 31, the structure 31
Different exposures to the wavelengths provided by

In the cell culture sequence defined in accordance with the present invention shown in FIGS.
In Cell Culture Disk ”, of course,
It is only an example that can be modified and supplemented. In particular, it is also possible to use other dimensions instead of those mentioned above, and to use other suitable materials in place of the materials used.

For example, instead of palladium, it is also possible to select another suitable special steel, organic substance or chemically fixed ligand. Also, other suitable, preferably transparent, hydrophobic polymers, metals, semiconductors or insulating materials can be used instead of polycarbonate. Also, instead of Teflon or polystyrene as the semi-permeable covering layer, other materials such as those commonly used for fully permeable, semi-permeable or non-permeable inner walls can be used. Agarose on silicon wafers has been forced to be used, especially because agarose is inert and is suppressed by cells during their growth. However, it is of course possible to use other materials with suitable properties for coating the silicon wafer. Finally, a transparent disk can be used instead of a silicon disk (quartz or other glass). Furthermore, disks made of, for example, polycarbonate or other polymers having low water absorption, heat resistance, dimensional stability, and high impact strength can be used. Various composite material structures are likewise modern, such as, for example, silicon honeycomb structures with fixed holes provided with quartz windows.

There are no limits on the possible applications of the arrangement defined according to the invention. In addition to the described application methods, it is also possible to use the aforementioned compact cellular structure discs, especially in space flight. For example, cells or cell cultures placed on a compact cell culture disc defined according to the present invention can be used to study the effects of, for example, weightlessness, cosmic rays, etc. on individual cells during their culture. It is also possible to expose to the conditions of.

[0045]

What is essential is that, when selecting various materials, it is possible to obtain an arrangement for culturing cells, whereby a large number of microchambers can be isolated from one another, preferably by half. The permeable inner wall is to be sealed from the surroundings and to be able to be viewed through a suitable window opening.

[Brief description of the drawings]

1 (a) is a plan view of an example of a disk-shaped arrangement according to the present invention, and FIGS. 1 (b) and 1 (c) are partially enlarged views of the arrangement of FIG. 1 (a). is there.

FIG. 2 is a cross-sectional view of the individual elements or components used in various exemplary cases in a schematic and enlarged view of a sandwich arrangement according to the invention.

FIG. 3 is a cross-sectional view of a variation of an embodiment of the present invention that can be arranged in an assembled and explosively deployed state.

FIG. 4 is a cross-sectional view of another embodiment variant of the arrangement according to the invention in a cross-sectional view of the assembled and explosively deployed depiction.

FIG. 5 is a sectional view of another embodiment variant of the arrangement according to the invention, newly assembled and exploded.

FIG. 6 is a schematic depiction of the in-situ measurement method possible with the arrangement according to the invention.

FIG. 7 is a schematic depiction of a cross section of another measuring method of the invention with a dead substance and a coating arrangement.

FIG. 8 is a cross-sectional view of a variant of an embodiment of the arrangement according to the invention, depicted schematically.

FIG. 9 is a cross-sectional view of another embodiment variant of a magnetic field-held arrangement according to the invention, depicted schematically.

10 (A) is a plan view of a disk-shaped circular array according to the present invention, and FIG. 10 (B) is a modification of another embodiment formed similar to a compact disk. It is a top view of a body.

FIG. 11: Possible applications of the arrangement according to the invention for investigating the biological effects of various radiation types on an object extracted as a function of coordinates in a sample system, depicted graphically in perspective. FIG.

[Explanation of symbols]

 Reference Signs List 1 disk 5 semi-permeable inner wall 6 matrix 7 silicon disk 8 disk 9 magnetic disk 11 container 17, 18 microchamber 19 notch

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[Procedure amendment]

[Submission date] July 15, 1997

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Fig. 1

[Correction method] Change

[Correction contents]

FIG. 1 shows an example of a disk-shaped arrangement according to the present invention and its arrangement
It is a top view which shows these partial expansion parts .

Claims (20)

[Claims] 1. A multi-chamber or pit (17) covered with a preferably transparent thin film, which is isolated from one another and is sealable from the surroundings by means of a selectably defined permeable inner wall (5). An array for culturing cells and / or other biological samples. 2. A microchamber or an inner wall (2)
2. The arrangement according to claim 1, wherein the pits (17) closable with 5) are arranged on a disc-shaped, flat, at least substantially smooth cradle (7). 3. The microchamber or the pit that can be covered by an inner wall is formed of a sandwich structure of congruent disc-shaped or thin-film elements that are adjusted in front of and behind, or are adapted to be opposite to each other. 3. The arrangement according to claim 2, wherein the one or more disks or membranes to be formed have a honeycomb-like or matrix-like structure with small holes (17). 4. The method as claimed in claim 2, wherein the pedestal is formed by a thin, plane-parallel, preferably smooth, optically advanced and uniform quality disk. The sequence described in the section. 5. At least a part of the chamber or pit (17) is formed by a honeycomb-shaped lattice structure or matrix (6) mounted on and securely connected to the cradle (7). Preferably, the small chamber (17) or the hollow opening of the lattice opposed to the cradle (7) can be covered or sealed, preferably by using a semi-permeable membrane (5) or a thin film. 5. The sequence according to claim 1, wherein 6. The individual chamber or pit (17) is finished with high precision to a circular shape having a diameter of about 0.2 to 5 mm,
6. The method according to claim 1, wherein at least a part of the chamber is arranged in the form of a honeycomb structure.
The sequence described in the section. 7. The cradle (7) is covered without cell affinity, preferably utilizing a transparent gel or polymer layer,
7. The arrangement according to claim 2, wherein pits (17) are arranged thereon. 8. A spot formed of a thin layer suitable for culturing cells, which is formed in the form of a dot or an island, is arranged on the bottom of a chamber mounted on a cradle or a gel layer or a polymer layer. The sequence according to claim 1, wherein the sequence is characterized by the fact that: 9. The grid structure or small chamber wall (15)
9. The arrangement according to claim 1, wherein the arrangement comprises a rigid, dimensionally stable material, in particular polycarbonate, silicon, a metallic material, a ceramic material or a composite material. 10. A coating, preferably semi-permeable, comprising:
Having selectable permeability properties, such as Teflon,
10. The arrangement according to claim 1, wherein the arrangement comprises a polymer material such as polystyrene or polycarbonate. 11. A hole (7) in the center for mounting a disc on a holding device of a microscope, measuring instrument or processing instrument.
11. The method as claimed in claim 3, wherein the circular disk having a diameter of about 5 to 20 cm.
The sequence described in the section. 12. The arrangement according to claim 1, wherein the arrangement or the structure is held in a sandwich by means of an arrangement of magnets. 13. To carry out a measurement on an array arranged in a container, the array is provided with a light-transmitting opening on one or both sides, for example a container such as a special steel box.
13. The arrangement according to claim 1, which is arranged in 1). 14. A method of producing an array for culturing cells or other biological samples, comprising the steps of: opening as hollow or small holes for individual cells or biological samples, ie for forming micro-laboratory or pits. Forming a lattice-like or honeycomb-like structure (6), including the cultured culture space (17); attaching and connecting the lattice-like or honeycomb-like structure on the semi-permeable membrane (5) or the coating layer; Nutrient substances, serum, active substances and / or other liquid or soluble or dissolvable components are filled in individual hollows or pores (17) sealed downward by a semi-permeable membrane or coating, A cell (23) or a biological sample is injected into the hollow or small hole or small chamber (17), and the small hole or hollow is semi-permeable thin film or coating layer (5).
Mounting a disc-shaped flat plate (7) as a pedestal for sealing against the above, and rotating the formed array by 180 °. 15. The method according to claim 14, wherein the semi-transparent inner wall having a honeycomb-like or lattice-like structure can be reliably connected using a UV-curable material. 16. The disk-shaped receiving base (7) is made of silicon,
It is made of quartz or glass and subsequently coated with a cell-friendly, preferably transparent gel or polymer layer, preferably an agarose layer (17), and finally the hollow or small structure (15). A point- or island-shaped cell-affinity spot (14) which is geometrically coincident with the hole (11) is provided, each time followed by the fact that said spot is essentially located at the center of the hollow or stoma. 16. The method according to claim 14 or 15, wherein the cradle is jointly connected to the structure. 17. The method according to claim 14, wherein the cradle and the structure are securely connected to one another using a fatty substance such as silicone grease. . 18. A structure (15), wherein a coating layer (25) is mounted on a metal plate or foil provided with small holes, said small holes geometrically corresponding to hollow or small holes in the structure. And following mounting of the cradle (23), the magnet is mounted on the cradle on the opposite side to the structure to retain the alignment as a result of tensioning the metal plate or foil by the magnet; 17. The method according to claim 14, wherein the array is finally rotated by 180 [deg.]. 19. The arrangement or chamber structure is disassembled so that the cultured material can be exposed and killed, and the killed material is measured using photon emission or thermal neutrons. Claims 1 to 1
3. A measurement method for performing measurement with the sequence according to any one of 3. 20. The sequence or compartment structure is disassembled such that the culture material is exposed for biochemical or molecular biological research methods such as electrophoresis or other separation methods. A measurement method for performing measurement with the sequence according to any one of claims 1 to 13.