WO2014000722A1 - Dispositif de marquage biochimique de tissus et de cultures cellulaires et utilisation dudit dispositif - Google Patents

Dispositif de marquage biochimique de tissus et de cultures cellulaires et utilisation dudit dispositif Download PDF

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Publication number
WO2014000722A1
WO2014000722A1 PCT/DE2013/000310 DE2013000310W WO2014000722A1 WO 2014000722 A1 WO2014000722 A1 WO 2014000722A1 DE 2013000310 W DE2013000310 W DE 2013000310W WO 2014000722 A1 WO2014000722 A1 WO 2014000722A1
Authority
WO
WIPO (PCT)
Prior art keywords
tub
trough
cell cultures
tissues
tilting
Prior art date
Application number
PCT/DE2013/000310
Other languages
German (de)
English (en)
Inventor
Markus Cremer
Karl-Heinz Beyer
Original Assignee
Forschungszentrum Jülich 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 Jülich GmbH filed Critical Forschungszentrum Jülich GmbH
Publication of WO2014000722A1 publication Critical patent/WO2014000722A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G01N1/312—Apparatus therefor for samples mounted on planar substrates

Definitions

  • the invention relates to a device for biochemical labeling of tissues and cell cultures and their use.
  • 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.
  • 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.
  • 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.
  • the lines are each in connection with storage containers, which store liquids for the respective process steps.
  • storage containers which store liquids for the respective process steps.
  • n reservoirs which receive the necessary solutions, n being able to assume, for example, 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.
  • 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.
  • the pumps are not required, but advantageous because it can be well dosed. Through the dosage, currents in the tub can be better controlled.
  • 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.
  • cover plate over the slides that can prevent evaporation of liquid.
  • 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.
  • 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.
  • 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.
  • 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 can be, for example, a plate or a grid on which the samples are located and which are connected with cables or chains which can be conveyed up and down by means of a motor via a thread.
  • 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.
  • 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.
  • 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 one Layer thickness in one to two-digit ⁇ range, for example, brain slices act.
  • large brain slices such as cuts of human or monkey brains can be introduced into the tub.
  • smaller brain slices such as mice, can also be used as the examination object.
  • the tissue samples are then in accordance with the Test course with changing liquids from the reservoirs acted upon. 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.
  • 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.
  • the tissue to be marked can be spared, since the liquids do not strike the tissue frontally.
  • 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.
  • the liquid is discharged via the liquid outlet.
  • the tilting device is raised at an angle to the horizontal and opened a possibly existing valve at the drain.
  • 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.
  • temperature ranges between see 4 ° C and 60 ° C, especially 4 ° C to 37 ° C, called.
  • Typical temperatures used are 22 ° C or 4 ° C.
  • 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.
  • a saving of radioactive ligands for example tritium [3 H] -labeled neurotransmitters
  • a saving of radioactive ligands for example tritium [3 H] -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.
  • the device can be used with all sub-combinations of the disclosed device features for the biochemical labeling of tissues and cell cultures.
  • the figures show the device according to the invention in a schematic form.
  • FIG. 5 shows a device according to the invention with lifting device
  • FIG. 6 shows a device according to the invention with lifting device before or after
  • FIG. 1 shows a device according to the invention.
  • reference numeral 1 denotes a trough in which slides 2, 2a, 2b are received, on which are located
  • the trough 1 is located on a means for tilting the trough 3. Between the means for tilting the trough 3 and the trough 1, a tempering 4 is positioned, on which the trough 1 is.
  • the tempering 4 is connected via lines 5, 5 a with thermostats 6, 6a in combination, which keeps the tempering 4 isothermally by means of a liquid circuit.
  • the tub 1 is connected via lines 7, 7a, 7b, 7c with reservoirs 8, 8a, 8b, 8c in combination, which supply them with necessary for the marking liquids.
  • pumps 9, 9a, 9b, 9c are mounted, which allow a supply of the liquid from the reservoirs 8, 8a, 8b, 8c.
  • the means for tilting the tub 3, the thermostats 6, 6a, and the pumps 9, 9a, 9b, 9c are connected to a control unit 10 in connection, which deliver control signals for the respective process steps.
  • a control unit 10 On the tub 1 is a liquid outlet 11 with a line 12 which leads into a collecting container 13.
  • FIG. 2 shows an inflow channel 18, via which the liquids from the storage containers 8 can be filled into the trough 1.
  • the bold line 19 indicates a flowed liquid covering the slides 2, 2a, 2b ....
  • FIG. 4 shows how the liquid in the trough 1 is discharged through the line 12 into the collecting container 13.
  • the means for tilting the tub 3 in the tilted state In this case, the means for tilting the tub 3 in the tilted state.
  • the fan 16 is in a lateral arrangement to the tub 1 and the plate for receiving slides 14 is located in a lifting device 20 with lifting means 21, which are in communication with a motor 22.
  • FIG. 6 shows the device according to FIG. 5 in a position in which the plate for picking up microscope slides 14 is lifted out by means of the lifting device 20.
  • Step 1 slides 2. 2a, 2b .... with cuts are placed in the tub 1.
  • Step 2 Water bath (tempering 4) is turned on and heats the tub 1 by means of the thermostat 6 a to room temperature (22 ° C).
  • Step 3 Pump 9 is turned on and pumps buffer (VIK) from the reservoir 8 to 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 passes through the liquid drain 1 1 and the hose 12 into the collecting container thirteenth
  • Step 6 The means for tilting the tub 3 moves back again.
  • Step 7 The pump 9a (HIK) is turned on and pumps buffer (HIK) from reservoir 8a to the cuts.
  • the inlet channel 18 swirls the buffer, so that no liquid jet reaches the cuts.
  • Step 8 The set incubation time runs, eg 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 overflows the liquid drain 1 1 out.
  • Step 10 The means for tilting the tub 3 moves back again. Start / end of the washing steps:
  • Step 1 1 The water bath 6a is turned on and heats the tub 1 by means of the thermostat 6b to 4 ° C.
  • Step 12 The wash buffer supply pump 9b is turned on and pumps (pre-cooled) buffer onto the cuts.
  • the inlet channel 18 swirls the buffer, so that no liquid jet reaches the cuts.
  • Step 13 The set incubation time is running, e.g. 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 goes beyond the liquid drain 1 1 out.
  • Step 15 The means for tilting the tub 3 moves back again.
  • Step: 16 to 19: Repeat from step 1 1-15 > 2nd wash step.
  • Step 20 Step 12: The pump 9c (distilled water) is turned 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 running, e.g. 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 goes beyond the liquid outlet 11.
  • Step 23 Acoustic signal, so that the tub 1 can be placed with the cuts under the fan 16.
  • the cover plate 15 of the tub can also be raised by motor control and the fan is controlled directly. Operation of the control unit

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un dispositif de marquage biochimique de tissus et de cultures cellulaires, ainsi que son utilisation. Le dispositif selon l'invention comprend une cuvette (1), destinée à recevoir des coupes tissulaires ou des cultures cellulaires, qui est posée sur des moyens d'inclinaison de la cuvette (3) et qui est reliée par l'intermédiaire de conduites d'alimentation (7) à des réservoirs (8) dans lesquels des solutions de marquage biochimique des tissus et des cultures cellulaires peuvent être prélevées. Les solutions sont introduites dans la cuvette (1) dans un ordre prédéfini au moyen d'une commande (10) afin d'être déposées sur les tissus ou les cultures cellulaires. Après le dépôt d'une solution, la cuvette (1) est inclinée et vidée puis la solution suivante est introduite dans la cuvette (1).
PCT/DE2013/000310 2012-06-30 2013-06-07 Dispositif de marquage biochimique de tissus et de cultures cellulaires et utilisation dudit dispositif WO2014000722A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102012013126.7 2012-06-30
DE201210013126 DE102012013126A1 (de) 2012-06-30 2012-06-30 Vorrichtung zur biochemischen Markierung von Geweben und Zellkulturen und deren Verwendung

Publications (1)

Publication Number Publication Date
WO2014000722A1 true WO2014000722A1 (fr) 2014-01-03

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PCT/DE2013/000310 WO2014000722A1 (fr) 2012-06-30 2013-06-07 Dispositif de marquage biochimique de tissus et de cultures cellulaires et utilisation dudit dispositif

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WO (1) WO2014000722A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT202100018659A1 (it) * 2021-07-15 2023-01-15 Bioevo S R L Sistema per la colorazione di vetrini per osservazioni microscopiche, e relativo metodo

Citations (5)

* Cited by examiner, † Cited by third party
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
US5338358A (en) * 1992-04-06 1994-08-16 Kabushiki Kaisha Tiyoda Seisakusho Apparatus for dyeing tissues
US20050186114A1 (en) * 2002-04-15 2005-08-25 Kurt Reinhardt Automated high volume slide processing system
US20060169719A1 (en) * 2003-08-11 2006-08-03 Bui Xuan S Manifold assembly

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6544788B2 (en) * 2001-02-15 2003-04-08 Vijay Singh Disposable perfusion bioreactor for cell culture
US7300789B2 (en) * 2002-05-21 2007-11-27 L'oreal Bioreactor forming a rigid vessel
AU2008100675A4 (en) * 2008-07-23 2008-08-28 Pak Leong Lim Portable shaker and method for performing assays

Patent Citations (5)

* Cited by examiner, † Cited by third party
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
US5338358A (en) * 1992-04-06 1994-08-16 Kabushiki Kaisha Tiyoda Seisakusho Apparatus for dyeing tissues
US20050186114A1 (en) * 2002-04-15 2005-08-25 Kurt Reinhardt Automated high volume slide processing system
US20060169719A1 (en) * 2003-08-11 2006-08-03 Bui Xuan S Manifold assembly

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