DE102012013126A1 - Device for biochemical labeling of tissues and cell cultures and their use - Google Patents

Device for biochemical labeling of tissues and cell cultures and their use

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Publication number
DE102012013126A1
DE102012013126A1 DE201210013126 DE102012013126A DE102012013126A1 DE 102012013126 A1 DE102012013126 A1 DE 102012013126A1 DE 201210013126 DE201210013126 DE 201210013126 DE 102012013126 A DE102012013126 A DE 102012013126A DE 102012013126 A1 DE102012013126 A1 DE 102012013126A1
Authority
DE
Germany
Prior art keywords
tub
device according
characterized
tissues
cell cultures
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
DE201210013126
Other languages
German (de)
Inventor
Markus Cremer
Karl-Heinz Beyer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Forschungszentrum Julich GmbH
Original Assignee
Forschungszentrum Julich GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Forschungszentrum Julich GmbH filed Critical Forschungszentrum Julich GmbH
Priority to DE201210013126 priority Critical patent/DE102012013126A1/en
Publication of DE102012013126A1 publication Critical patent/DE102012013126A1/en
Application status is Withdrawn legal-status Critical

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00Sampling; Preparing specimens for investigation
    • G01N1/28Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30Staining; Impregnating Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31Apparatus therefor
    • G01N1/312Apparatus therefor for samples mounted on planar substrates

Abstract

The invention relates to a device for biochemical labeling of tissues and cell cultures and their use.
The device according to the invention comprises a tub (1) for receiving tissue sections or cell cultures, which is located on means for tilting the tub (3) and which is connected via supply lines (7) with storage containers (8), from which solutions for biochemical labeling the tissues and cell cultures can be obtained. The solutions are directed in a predetermined order by means of a controller (10) into the trough (1) to charge the tissues or cell cultures. After exposure to a solution, the tub (1) is tilted and emptied. Thereafter, the next solution in the tub (1) is passed.

Description

  • The invention relates to a device for biochemical labeling of tissues and cell cultures and their use.
  • In medical and biological research methods for the biochemical labeling of tissues but also cell cultures are used in the prior art. Known methods are autoradiography, in situ hybridization or immunohistochemistry. In these procedures, tissues are labeled by different means. Thus, a radioactive labeling of neurotransmitters takes place, which bind to receptors or radioactive probes are used to label gene segments. Furthermore, with dye or radioactively labeled antibodies can be used. Applications are also found in histology. Basically, any tissue can be examined, particularly interesting is the investigation of brain slices.
  • In the methods used in the prior art, the tissue samples or the brain slices are applied to slides, which are usually mounted vertically in holding devices. In this case, several slides can be fixed with samples in a holder.
  • These samples are then placed, depending on the method used, in a specific sequence in different vessels with solutions that act on them. The solutions are, for example, buffer solutions, dye solutions or solutions which contain radioactive markers which are brought into contact with the tissue samples, for example brain slices. This work is done manually.
  • In the prior art approach, there is the difficulty that the actions performed by a person are bound to a strictly predetermined order and accuracy in performance, and that deviations in performance may occur in the context of error accuracy. Furthermore, this procedure requires large amounts of particularly valuable and expensive materials, which causes very high costs. For example, radioactive markers are high-priced substances, but also other substances necessary for the processes have to be produced in a complicated manner and are very expensive. There is therefore a need to carry out the processes as precisely reproducible as possible and to use expensive materials sparingly.
  • It is therefore the object of the invention to provide a device for carrying out biochemical markings of tissues, in particular of brain sections or cell cultures, with which the marking work can be carried out with high precision and reproducibility and the saving of expensive material, such as radioactive markers and other valuable solutions allows.
  • With the device according to the invention a highly accurate and reproducible performance of biochemical labeling experiments is possible, which is not dependent on the manual skill of the experimenter, and it will - compared to the manual processing - up to 60% of the cost of valuable materials, such as radioactive marker, antibody or Color solutions, compared to the manual process saved. The temperature conditions are optimized. Automatic control and logging is possible.
  • Starting from the preamble of claim 1, the object is achieved by the features specified in the characterizing part of claim 1.
  • In the following, the invention will be described in its general form.
  • The core of the device consists of a tub for receiving tissues, which is equipped with means for tilting the tub. The tissues are preferably on slides.
  • The means for tilting the tub may be a tilting table or a rack which receives the tub.
  • The means for tilting the tub are equipped with a motor which allows tilting of the tub.
  • Into the tub leads enter, which supply them with different liquids.
  • The lines are each in connection with storage containers, which store liquids for the respective process steps. In the device according to the invention, at least two storage containers are provided. One reservoir is filled with one solution for the mark and the other with a wash solution. Depending on the method chosen, there must be n reservoirs that receive the necessary solutions. For example, n can assume a value of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Accordingly, the same number n is provided on lines that connect the tub with the reservoirs.
  • In this case, valves are provided which allows regulation of the inflow of liquids. The valves may be positioned in the conduit, at the reservoirs and / or at the entrances to the trough.
  • In a preferred embodiment, there are pumps on the lines, which promote the solutions defined in the reservoirs.
  • If the storage containers are arranged above the tub, the pumps are not required, but advantageous because it can be well dosed. Through the dosage, currents in the tub can be better controlled.
  • Advantageously, in the position where the liquid supply lines enter the trough, there is a feed trough which receives the respective supplied liquid and allows a smooth supply of liquid to the slides so that the tissue sections are not damaged or compromised , For this purpose, it is advantageous if the inlet channel is arranged substantially perpendicular to the liquid flow, which is passed into the trough. It is preferably arranged to direct the liquid from the reservoirs into the side region of the trough so that the liquid flow does not run directly over the cuts and any shearing forces that may occur are reduced.
  • Furthermore, there may be a cover plate over the slides that can prevent evaporation of liquid.
  • On the tub, a liquid discharge is further attached, for example in the form of a drain valve, which is preferably located on the opposite side of the tub lines.
  • In a preferred embodiment, the tub is on a tempering, which is located on the means for tilting the tub. The tempering can also be integrated in the bottom of the tub. The tempering element may be a metal block or a metal plate. The tempering element can be equipped with cooling and heating coils, which are connected via lines with thermostats.
  • In this case, different thermostats can be operated independently of each other in the tempering, which are connected to different thermostats that keep different temperatures constant. The temperature control coils are connected via lines with valves to the respective thermostat, so that optionally different temperature control circuits can be activated. As a temperature control, for example, water, a water-glycerin mixture or commercially available coolant into consideration.
  • A cover plate, for example made of Plexiglas, which protects the tissue samples or sections from external influences and prevents evaporation of the liquids used during the exposure time, can be located above the tub.
  • Advantageously, a lifting device can be mounted above the trough, which allows the insertion and removal of tissues or slides with tissues, brain slices or cell cultures in order to protect the possibly unfixed examination material from damage by the liquid jet. The lifting device may be, for example, a plate or a grid on which the samples are located and which are connected to cables or chains which can be conveyed up and down by means of a motor via a thread.
  • Furthermore, the device according to the invention can be equipped with a blower or fan, which allows drying of the marked samples.
  • The means for tilting the tub, the valves that regulate the flow of liquids from the reservoirs, the drain valve and the thermostats or individual components from this group are connected to a controller, in which the respective timing of the influx of liquids from each container , the timing of the tilting of the tub, and the time course of the temperature of the heating element is programmed and the opening and closing of valves and the tilt angle of the tub, as well as the heating of the thermostats for the different temperature control circuits and / or opening or closing of Valves of the temperature control circuits or the drain valve controls. Thus, any desired method can be carried out automatically.
  • During operation, the storage containers are filled with the necessary liquids. These may be buffers, aqueous solutions, solutions of radiolabels, dyes and other liquids which may be contacted with the tissue or cell samples according to the time sequence.
  • Tissue samples are placed on microscope slides in the tub. The tissue samples may be any type of cryostat or paraffin / microtome sections, in particular a layer thickness in the range of one to two digits, for example brain sections. In particular, large brain slices, such as cuts of human or monkey brains can be introduced into the tub. However, smaller brain slices, such as mice, can also be used as the examination object. The tissue samples are then applied according to the preprogrammed course of the experiment with changing fluids from the reservoirs. This can be done by placing the samples in the tub and then opening valves for the supply of liquid from a reservoir so that the samples are flooded. The flooding can be done via a feed channel, which receives the liquid and the tub at the side areas supplies, so that the tissue sections are not affected by currents. Preferably, the inlet channel ends at a small distance from the wall of the tub, so that the liquid can run both laterally and over the front edge of the inlet channel into the tub. Through these measures, the tissue to be marked can be spared, since the liquids do not strike the tissue frontally.
  • Alternatively, the empty tub can be filled by opening a valve for the supply of a liquid from a reservoir, and then the slides with tissue or brain slices, which are located on a plate, can be lowered from above into the tub until with the liquid submitted are covered. This has the advantage that the surface of the tissue samples is not attacked by the flowing liquid.
  • After a predetermined exposure time, the liquid is discharged via the liquid outlet. For this purpose, the tilting device is raised at an angle to the horizontal and opened a possibly existing valve at the drain.
  • In an advantageous embodiment, the slides with the tissue samples are previously lifted out of the tub again. This has the advantage that the surface of the tissue samples is not attacked by the flowing liquid. Furthermore, the lifted tissue samples can be dried by a fan before being reinserted into the tub.
  • The process is then repeated by means of a control unit after the programmed time sequence with liquids from different storage containers. Various process steps can be carried out at different temperatures, as described for example in the special part of the description. By way of example, temperature ranges between 4 ° C. and 60 ° C., in particular 4 ° C. to 37 ° C., may be mentioned. Typical temperatures used are 22 ° C or 4 ° C.
  • During the various filling cycles, the temperature of the heating element can be changed by switching on and off the supply of various fluid circuits of the thermostats as preprogrammed.
  • By operating with the device according to the invention, a saving of radioactive ligands, for example tritium [3H] -labeled neurotransmitters, can be achieved by a reduced main incubation volume. The user-independent incubation steps lead to error-free compliance with times for different process sections. The temperature conditions are optimized and automatic control and logging of the process is possible.
  • According to the invention, the device can be used with all sub-combinations of the disclosed device features for the biochemical labeling of tissues and cell cultures.
  • Special description part:
  • In the following, the device according to the invention will be described by means of examples without these examples being interpreted restrictively.
  • The figures show the device according to the invention in a schematic form.
  • The figures show:
  • 1 : a device according to the invention
  • 2 : a device according to the invention in side view
  • 3 a device according to the invention during the filling of buffer liquid
  • 4 : a device according to the invention in tilted position
  • 5 a device according to the invention with lifting device
  • 6 a device according to the invention with a lifting device before or after the marking of samples.
  • In 1 a device according to the invention is shown. In her, the reference numeral 1 a tub, in the slide 2 . 2a . 2 B ..... are recorded on which tissue sections or cell cultures are located. The tub 1 is located on a means for tilting the tub 3 , Between the means for tilting the tub 3 and the tub 1 is a tempering element 4 positioned on which the tub 1 stands. The tempering element 4 stands over lines 5 . 5a with thermostats 6 . 6a in conjunction, the tempering 4 isothermal by means of a liquid circulation. The tub 1 stands over lines 7 . 7a . 7b . 7c with storage containers 8th . 8a . 8b . 8c in conjunction, they supply with necessary for marking liquids. On the lines 7 . 7a . 7b . 7c are pumps 9 . 9a . 9b . 9c attached, which is a supply of liquid from the reservoirs 8th . 8a . 8b . 8c enable. The means for tilting the tub 3 , the thermostats 6 . 6a , and the pumps 9 . 9a . 9b . 9c stand with a control unit 10 in connection, which deliver control signals for the respective process steps. At the tub 1 there is a liquid drain 11 with a line 12 placed in a container 13 leads.
  • In 2 the same device features have the same reference numerals. In her is in the tub 1 a plate for holding the slides 14 shown. Over the tub 1 there is a cover plate 15 , Above the tub is a fan 16 appropriate. The dashed lines indicate control lines 17 for tilting the means for tilting the tub 17a , for opening and closing the drain valve 17b , for regulating the thermostat 17c . 17d and for the opening and closing of the valves or pumps 17e and for the regulation of the supply of fluids 17f , The reference number 18 numbered an inlet channel.
  • In 3 becomes an inlet channel 18 represented over which the liquids from the supply containers 8th in the tub 1 can be filled. The bold line 19 refers to a stream of liquid containing the slides 2 . 2a . 2 B .... covered.
  • In 4 is shown as in the tub 1 located liquid through the line 12 in the collection container 13 is drained. This is the means for tilting the tub 3 in tilted condition.
  • In 5 is the fan 16 in lateral arrangement to the tub 1 and the plate for receiving slides 14 is in a lifting device 20 with lifting agents 21 that with a motor 22 keep in touch.
  • 6 shows the device according to 5 in a position where the plate is for holding slides 14 by means of the lifting device 20 is lifted out.
  • Example:
  • The operation of the device according to the invention is exemplified by the following procedure programmed in the controller.
  • Start / end of preincubation (VIK):
    • • Step 1: Slide 2 , 2a . 2 B .... with cuts are in the tub 1 placed.
    • Step 2: Water bath (tempering element 4 ) turns on and heats the tub 1 by means of the thermostat 6a to room temperature (22 ° C).
    • • Step 3: Pump 9 is switched on and pumps buffer (VIK) from the reservoir 8th on the cuts.
    • The inlet channel 18 swirls the buffer, so that no liquid jet reaches the cuts.
    • • Step 4: Set incubation time is running, eg. B. 15 min.
    • • Step 5: The means for tilting the tub 3 is turned on. The tub 1 with the cuts is tilted to 45 °. The VIK buffer runs over the liquid drain 11 and the hose 12 in the collection container 13 ,
    • • Step 6: The means for tilting the tub 3 goes back again.
  • Start / end of main incubation (HIK):
    • • Step 7: The pump 9a (HIK) is turned on and pumps buffer (HIK) from reservoir 8a on the cuts.
    • The inlet channel 18 swirls the buffer, so that no liquid jet reaches the cuts.
    • • Step 8: The set incubation time is running, eg. B. 60 min.
    • • Step 9: The means for tilting the tub 3 is turned on. The tub 1 with the cuts is tilted to 45 °. The HIK buffer runs over the liquid drain 11 out.
    • • Step 10: The means for tilting the tub 3 goes back again.
  • Start / end of the washing steps:
    • • Step 11: The water bath 6a turns on and heats the tub 1 by means of the thermostat 6b at 4 ° C.
    • • Step 12: The pump 9b for the supply of wash buffer is turned on and pumps (precooled) buffer on the sections.
    • The inlet channel 18 swirls the buffer, so that no liquid jet reaches the cuts.
    • • Step 13: The set incubation time is running, eg. B. 5 min.
    • • Step 14: The means for tilting the tub 3 is turned on. The tub 1 with the cuts is tilted to 45 °. The buffer runs over the liquid drain 11 out.
    • • Step 15: The means for tilting the tub 3 goes back again.
    • • Step: 16 to 19: Repeat from step 11-15 => 2nd washing step.
  • Start / end of the wash with dest. Water:
    • • Step 20: Step 12: The pump 9c (at least water) is switched on and pumps (precooled) dist. Water from container 8c on the cuts.
    • The inlet channel 18 swirls the water, so that no liquid jet reaches the cuts.
    • • Step 21: Set incubation time is running, eg. B. 2 sec.
    • • Step 22: The means for tilting the tub 3 is turned on. The tub 1 with the cuts is tilted to 45 °. The water runs over the liquid outlet 11 out.
    • • Step 23: Acoustic signal to allow the tub 1 with the cuts under the fan 16 can be made. Optionally, the cover plate 15 the tub can also be raised by motor control and the fan is controlled directly.
  • Operation of the control unit
    • • (control) impulses are delivered at certain times, eg. Eg "On => Start Engine".
    • • Modern components (eg laboratory pumps or tilting tables) already have interfaces that can be controlled or read out with the "Labview" software.
  • Here is an example of controlling the various components: Start: VIK End: VIK End: VIK Start: HIK End: HIK Start: washing Start: washing etc. pump 9 (VIK) X VIK buffer is running pump 9a (HIK) X HIK buffer goes in pump 9b (To wash) X Wash buffer runs in pump 9c (Dest.) Lift the tilting motor 3 X VIK buffer expires X HIK buffer expires Lower tilting motor 3 X tub back to starting position X tub back to starting position temperature 6b : 4 ° C X X temperature 6a : 22 ° C X X X X timeline 0 min 15 minutes 15 min 5 sec 15 min. 10 sec 75 min. 10 sec 75 min 15 sec 75 min 20 sec etc.

Claims (10)

  1. Device for biochemical labeling of tissues or cell cultures with - a tub ( 1 ), - means for tilting the tub ( 3 ), - a liquid discharge ( 11 ), attached to the tub ( 1 ), - at least two storage containers ( 8th . 8a ), - Cables ( 7 . 7a ), which are the reservoirs ( 8th . 8a ) and the tub 1 connect, - pumps and / or valves ( 9 . 9a ,), which regulates the flow of liquid from the reservoirs ( 8th . 8a ) to the tub ( 1 ) and with a control unit ( 10 ), which the means for tilting the tub ( 3 ) and the pumps and / or valves ( 9 . 9a ).
  2. Apparatus according to claim 1, characterized in that the tub ( 1 ) with a tempering element ( 4 ) is in contact or contains it.
  3. Device according to claim 2, characterized in that the tempering element ( 4 ) with at least one thermostat ( 6 ).
  4. Device according to one of claims 1 to 3, characterized in that at the position of the trough ( 1 ) into which the supply lines ( 7 . 7a ...), an inlet channel ( 18 ) is attached.
  5. Device according to one of claims 1 to 4, characterized in that it comprises a plate for receiving slides ( 14 ) owns.
  6. Apparatus according to claim 5, characterized in that it comprises a lifting device ( 20 ) with lifting means ( 21 ), which holds the plate for holding the slides ( 14 ) can lift.
  7. Device according to one of claims 1 to 6, characterized in that above the tub ( 1 ) a cover plate ( 15 ) is located.
  8. Device according to one of claims 1 to 7, characterized in that it has a fan ( 16 ), which allows drying of samples.
  9. Device according to one of claims 1 to 8, characterized in that the liquid discharge ( 11 ) on the side of the tub ( 1 ), the entry of the lines ( 7 . 7a ) is opposite.
  10. Use of the device according to one of claims 1 to 9 for biochemical labeling of tissues and cell cultures.
DE201210013126 2012-06-30 2012-06-30 Device for biochemical labeling of tissues and cell cultures and their use Withdrawn DE102012013126A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DE201210013126 DE102012013126A1 (en) 2012-06-30 2012-06-30 Device for biochemical labeling of tissues and cell cultures and their use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE201210013126 DE102012013126A1 (en) 2012-06-30 2012-06-30 Device for biochemical labeling of tissues and cell cultures and their use
PCT/DE2013/000310 WO2014000722A1 (en) 2012-06-30 2013-06-07 Device for biochemically marking tissues and cell cultures and the use thereof

Publications (1)

Publication Number Publication Date
DE102012013126A1 true DE102012013126A1 (en) 2014-01-02

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DE201210013126 Withdrawn DE102012013126A1 (en) 2012-06-30 2012-06-30 Device for biochemical labeling of tissues and cell cultures and their use

Country Status (2)

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DE (1) DE102012013126A1 (en)
WO (1) WO2014000722A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
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US20030036192A1 (en) * 2001-02-15 2003-02-20 Vijay Singh Disposable perfusion bioreactor for cell culture
US20040029267A1 (en) * 2002-05-21 2004-02-12 L'oreal Bioreactor forming a rigid vessel
DE202009009980U1 (en) * 2008-07-23 2009-11-19 Lim, Pak Leong Portable shaker for performing assays and biomolecular reactions

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Publication number Priority date Publication date Assignee Title
US4084541A (en) * 1975-04-22 1978-04-18 Olympus Optical Co., Ltd. Dyeing and decolorization apparatus for use in a blood serum analyzer of an electrophoretic type
US4911098A (en) * 1987-12-28 1990-03-27 Shiraimatsu & Co., Ltd. Automatic straining apparatus for slide specimens
JP3320444B2 (en) * 1992-04-06 2002-09-03 株式会社千代田製作所 Nozzle cleaning equipment for dyeing equipment
US7468161B2 (en) * 2002-04-15 2008-12-23 Ventana Medical Systems, Inc. Automated high volume slide processing system
US7744817B2 (en) * 2003-08-11 2010-06-29 Sakura Finetek U.S.A., Inc. Manifold assembly

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030036192A1 (en) * 2001-02-15 2003-02-20 Vijay Singh Disposable perfusion bioreactor for cell culture
US20040029267A1 (en) * 2002-05-21 2004-02-12 L'oreal Bioreactor forming a rigid vessel
DE202009009980U1 (en) * 2008-07-23 2009-11-19 Lim, Pak Leong Portable shaker for performing assays and biomolecular reactions

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Publication number Publication date
WO2014000722A1 (en) 2014-01-03

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Effective date: 20141021