WO2007054159A1 - Dispositif destine a l'obtention de suspensions cellulaires avec incorporation ulterieure d'additifs et procede associe - Google Patents

Dispositif destine a l'obtention de suspensions cellulaires avec incorporation ulterieure d'additifs et procede associe Download PDF

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Publication number
WO2007054159A1
WO2007054159A1 PCT/EP2006/009386 EP2006009386W WO2007054159A1 WO 2007054159 A1 WO2007054159 A1 WO 2007054159A1 EP 2006009386 W EP2006009386 W EP 2006009386W WO 2007054159 A1 WO2007054159 A1 WO 2007054159A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
supernatant
punch
hollow punch
protective agent
Prior art date
Application number
PCT/EP2006/009386
Other languages
German (de)
English (en)
Inventor
Thomas Montag-Lessing
Ingo Spreitzer
Bettina Löschner
Bettina Ziegele
Original Assignee
Paul-Ehrlich-Institut Bundesamt Für Sera Und Impfstoffe
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 Paul-Ehrlich-Institut Bundesamt Für Sera Und Impfstoffe filed Critical Paul-Ehrlich-Institut Bundesamt Für Sera Und Impfstoffe
Publication of WO2007054159A1 publication Critical patent/WO2007054159A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation

Definitions

  • the present invention relates to a device for obtaining cell suspensions with subsequent admixture of additives, in particular protective agents for cryopreservation.
  • the device of the invention eliminates the need for cell isolations and other expensive and potentially contaminating treatment steps.
  • the present invention further relates to a method of obtaining cell samples using the device.
  • PBMCs mononuclear cells
  • WO 2005-089837 describes a two-component prefilled syringe.
  • US 5,522,804 describes a syringe for aspiration, mixing and injection of substances.
  • the syringe contains a chamber integrated into its piston for substances that can be injected after injection of a first substance. It is an object of the present invention to provide a device which facilitates facilitated, safe and thus improved sample preparation. It should thereby be developed a device with which in particular individual blood samples can be obtained and mixed with additives, so that the samples without further processing directly in the device with the least possible effort and at the lowest possible cost cryopreserved and transported (eg for the clinical Testing vaccines against pathogens defended by cell-mediated immunity to determine the individual immune status of patients).
  • a device for preparing a blood sample which has a sample chamber (12) with an inlet (1), a movable plunger (3) with a stamp plate with pores (2) arranged inside the sample chamber. , and a second chamber (5), within the sample chamber (12), for sample conditioning means connected to the sample chamber (12) via a closable inlet (8).
  • a device has been developed in which, in particular, individual blood samples can be obtained and admixed with additives, so that the samples can be frozen directly in the device according to the invention without further processing.
  • the monocytes and lymphocytes of the samples retain their function and are not significantly activated.
  • the cryopreservation and the transport are carried out according to the invention without liquid nitrogen. This makes it possible to obtain individualized blood samples (for clinical vaccine testing, for example) with very little financial and technical effort.
  • the principle is also applicable to other cell suspensions.
  • the second chamber is formed by a within the sample chamber (12) lying movable hollow punch (7), in particular by a sealing washer (6).
  • the sealing disk (6) is entrained by suitable surface design (eg roughness) of the overflow channels (8).
  • the gasket can be connected via a groove with the hollow punch (7).
  • the device is provided with a multi-step fixation principle for the hollow die in the lower position, as will be explained below.
  • a device according to the invention is characterized in that the closable inlet (8) of the second chamber (5) can be opened by a rotary and / or piercing mechanism by means of the hollow punch (7).
  • the protective means (5) passes through the transfer channels (8) into the supernatant (for example plasma).
  • the gasket (6) is still moveable when a little more pulling is applied.
  • a connection can be made between the hollow die and sealing washer (e.g., via a groove) which can be dissolved (e.g., by spinning) prior to mixing the supernatant with the treating agent (e.g. By rotating the device several times overhead, uniform mixing of the supernatant with the treating agent (e.g., preservative) is achieved.
  • this is thus characterized in that the sealing disc (6) via a groove with the hollow punch (7) is detachably connected.
  • the closable inlet (8) comprises overflow channels (8) with a guide for the sealing disc (6).
  • this is characterized in that the punch (3) is movably mounted within the hollow punch (7). It is preferred that the punch (3) is guided by a sealing ring (4).
  • this is characterized in that the punch (3) and the hollow punch (7) by a releasable connection clip (9) are interconnected.
  • the connection clip (9) provides for the defined simultaneous retraction of both punches (3 and 7), but if necessary removed from the punch (3) and from the hollow punch (7).
  • a rotatable punch may be provided with a groove connection or other locking, which is then released accordingly.
  • this is characterized in that a stop and / or a corresponding marking is provided for the hollow punch (7) within the first sample space (12).
  • a stop is preferably provided for the hollow punch (7), which guarantees the stop of both punches in such a way that no protective means (5) is pressed into the sample space.
  • the hollow die can be fixed in this position by turning (for example by 90 °).
  • the device contains a mark for stopping the stamper (e.g., for blood at about 50% of the total sample volume) so as not to subject the cells to any mechanical pressure.
  • Turbulence can lead to a first breakthrough of protective agent through the overflow channels as well as to mixing with supernatant (for example plasma), which is not disadvantageous, since in the next step a thorough mixing of protective agent and supernatant takes place.
  • a device is preferred in which, by further turning (for example 90 °) of the hollow punch (7), it is released from the first holder and can be pulled out of the device until it stops against the sealing disk (6).
  • the protective means (5) passes through the transfer channels (8) into the supernatant (for example plasma).
  • the gasket (6) is still moveable when a little more pulling is applied.
  • a connection can be made between the hollow die and sealing washer (e.g., via a groove) which can be loosened (e.g., by rotation) prior to mixing the supernatant with the preservative.
  • this is characterized in that the hollow punch (7) within the first sample space (12), in particular by a rotation, is fixable to the stop.
  • the inlet (1) has a screw cap with a thread. This facilitates both the filling of the sample, as well as the closing and further storage.
  • the porous stamp plate (2) has a pore size of between 500 nm to 10 ⁇ m, preferably from 1 ⁇ m to 7 ⁇ m, most preferably from 2 ⁇ m to 5 has ⁇ m.
  • the pore size is chosen (e.g., 2-5 ⁇ m) so that smooth passage of the liquid takes place without clogging of the stamp plate, but prevents passage of cells.
  • protective agent-containing supernatant and cells are mixed.
  • the supernatant as a liquid penetrate the pores of the stamp plate and there is automatically optimal mixing of protective agent-containing supernatant and cell suspension.
  • the now protective agent-containing cell suspension can be frozen directly in the device. After storage and thawing, the cell suspension can be recovered from the device by pressure on the punches.
  • this is characterized in that the punches (3, 7) are provided with a predetermined breaking point and / or a corresponding marking or the grips of the punches (3, 7) are detachably provided.
  • the device can thus be provided with a predetermined breaking point for the stamp, which can be canceled for storage.
  • the device has on the connection side a screw cap, which is removed to recover the cell suspension.
  • the sample conditioning agent is selected from a cryopreservative, especially DMSO, hydroxyethyl starch (HES), such as final 10% or 20% DMSO in PBS or other aqueous buffer.
  • a cryopreservative especially DMSO, hydroxyethyl starch (HES), such as final 10% or 20% DMSO in PBS or other aqueous buffer.
  • this is completely pyrogen-free so as not to interfere with the measurements.
  • a further aspect of the present invention then relates to a method for preparing and freezing a blood sample, comprising the steps of a) providing a device according to the invention as above, b) providing a blood sample to be frozen, c) drawing the blood sample into the device from a) up to d) closing the inlet of the device, e) removing the connecting clip (9), f) separating cells and supernatant by pushing in the stamp (3), g) mixing protective agent and supernatant, h) mixing protective agent-containing supernatant and cells, and i) freezing the protective agent-containing cell suspension.
  • the supernatant is plasma. Even more preferred is a method according to the invention, wherein a uniform mixing of the supernatant with the protective agent is carried out by multiple overhead rotation of the device.
  • a final aspect of the present invention then relates to the use of a device according to the invention for preparing and freezing a blood sample, as stated above.
  • the e.g. Syringe contains all the necessary means to prepare the spot for a freezing process.
  • no further expensive devices except a -80 ° refrigerator or other suitable freezer at temperatures of -100 ° C or higher are required.
  • Blood samples can be obtained directly in the device according to the invention with minimal effort, the additives can be mixed gently and the samples are frozen. Further, cryopreservation can be accomplished without the use of liquid nitrogen (e.g., in the -80 ° C freezer or on dry ice). The principle can also be used for other cell suspensions.
  • Figure 1A to C show schematically a preferred apparatus of the present invention and its use in sampling and sealing of the apparatus.
  • Figures 2 A to C show schematically the preferred apparatus of the present invention of Figure 1 and its use in staple removal, filtration and mixing of the cell-free supernatant with sample conditioning agent (preservative).
  • Figure 3A shows schematically a preferred apparatus of the present invention of Figures 1 and 2 and their use in mixing the whole sample with the sample conditioning agent (preservative). Not shown are further steps of freezing at between -7O 0 C and -100 ° C without liquid nitrogen in a refrigerator or on dry ice.
  • FIG. 4 shows a further preferred device of the present invention, in which the second chamber (5) is opened by means of a piercing device (13).
  • the second chamber (5) is separated from the sample space (12) in this case by a membrane (14) (A).
  • A a membrane
  • the membrane (14) or other partition is pierced or pushed up by the piercing device (13) while filtration is taking place (B).
  • the protective agent is released into the sample chamber (12) and mixed with retraction of the punch (3) with the plasma.
  • Step 1 Sampling ( Figure IA and B)
  • the sampling (eg blood with anticoagulants) is carried out as with a conventional syringe.
  • Sealing ring (4) and movable sealing washer (6) guarantee the necessary negative pressure for mounting the sample. If these parts were not present, there would be no negative pressure in the sample chamber (12) when retracting the stamp, since air would pass through the porous stamp plate (2) into the sample chamber (12).
  • the sealing disc (6) is pulled along by suitable surface design (eg roughness) of the overflow channels (8).
  • the gasket can be connected via a groove with the hollow punch (7).
  • step 5 mixing the supernatant with the protective agent
  • the connection between the sealing washer and the hollow punch would then be released (eg by turning the punch).
  • Step 2 Remove the cannula, close the device ( Figure IC)
  • the device After removing the Luer-Lock cannula (10), the device is closed with a commercially available Luer-Lock cover (1).
  • the closure is a prerequisite for step 4 (see there), but also serves to protect the sample.
  • Step 3 Remove clamp ( Figure 2A)
  • connection clip (9) is removed from the punch (3) and the hollow punch (7).
  • Step 4 Separate cells from the supernatant ( Figure 2B)
  • the punch (3) By pushing in the punch (3), cells and supernatants (e.g., blood and plasma) are separated because the liquid can pass through the porous stamp plate (2) but not the cells.
  • the pore size is chosen (e.g., 2-5 ⁇ m) so that smooth passage of the liquid takes place without clogging of the stamp plate, but prevents passage of cells.
  • the device contains a mark for stopping the stamper (e.g., for blood at about 50% of the total sample volume) so as not to subject the cells to any mechanical pressure.
  • the hollow punch remains fixed in its intermediate position (see step 1). Turbulence can lead to a first breakthrough of protective agent through the overflow channels as well as to mixing with supernatant (for example plasma), which is not disadvantageous, since in the next step a thorough mixing of protective agent and supernatant takes place.
  • Step 5 Mix supernatant with preservative ( Figure 2C)
  • the protective means (5) passes through the overflow channels (8) in the supernatant (eg plasma). Due to the design of the surface (eg roughness, no firm connection to the hollow punch) of the overflow channels (8), the sealing disc (6) is still movable, if a little stronger train is exercised. Alternatively, a connection can be made between the hollow punch and the sealing washer (eg via a groove), which can be loosened before mixing the supernatant with the protective agent (eg by turning). By repeatedly turning the device upside down, a uniform mixing of the supernatant with the protective agent is achieved. The amount of protective agent is chosen so that the cells are optimally protected during cryopreservation.
  • Step 6 Blend Protecting Agent-containing Supernatant with Cells ( Figure 3A)
  • protective agent-containing supernatant and cells are mixed.
  • the supernatant as a liquid can penetrate the pores of the stamp plate.
  • Optimal mixing of protective agent-containing supernatant and cell suspension takes place automatically.
  • the now protective agent-containing cell suspension can be frozen directly in the device.
  • the cell suspension can be recovered from the device by pressure on the punches.
  • the device may be provided with a predetermined breaking point for the stamp, which can be canceled for storage. In this case, it is advantageous if the device has a screw cap on the connection side, which is removed for obtaining the cell suspension.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Environmental Sciences (AREA)
  • Dentistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Mechanical Engineering (AREA)
  • Hematology (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un dispositif d’obtention de suspensions cellulaires avec incorporation ultérieure d’additifs, notamment d’agents de protection pour cryoconservation. Le dispositif selon l’invention permet notamment de supprimer la nécessité d'une centrifugation et autres étapes de préparation coûteuses et possiblement contaminantes. La présente invention concerne également un procédé d’obtention d’échantillons cellulaires utilisant le dispositif selon l'invention.
PCT/EP2006/009386 2005-11-09 2006-09-27 Dispositif destine a l'obtention de suspensions cellulaires avec incorporation ulterieure d'additifs et procede associe WO2007054159A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200520017529 DE202005017529U1 (de) 2005-11-09 2005-11-09 Vorrichtung zur Gewinnung von Zellsuspensionen unter anschließender Beimischung von Zusatzstoffen
DE202005017529.3 2005-11-09

Publications (1)

Publication Number Publication Date
WO2007054159A1 true WO2007054159A1 (fr) 2007-05-18

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Application Number Title Priority Date Filing Date
PCT/EP2006/009386 WO2007054159A1 (fr) 2005-11-09 2006-09-27 Dispositif destine a l'obtention de suspensions cellulaires avec incorporation ulterieure d'additifs et procede associe

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DE (1) DE202005017529U1 (fr)
WO (1) WO2007054159A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107980764A (zh) * 2017-11-28 2018-05-04 上海原能细胞生物低温设备有限公司 一种低温中转箱

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4132480A1 (de) * 1991-09-30 1993-04-08 Kabe Labortechnik Gmbh Vorrichtung zur blutentnahme
US5522804A (en) * 1994-02-15 1996-06-04 Lynn; Lawrence A. Aspiration, mixing, and injection syringe
DE19520883A1 (de) * 1995-06-08 1996-12-12 Szibor Reinhard Prof Dr Vorrichtung zur Blutentnahme mit einem zweikammrigen System
WO2000062061A1 (fr) * 1999-04-12 2000-10-19 Institut für Chemo- und Biosensorik Münster E.V. Dispositif et procede a utiliser lors de la realisation de tests relatifs a des systemes recepteur-ligand et de tests d'affinite
WO2002067778A2 (fr) * 2001-02-26 2002-09-06 Ben-Ami Ballin Seringue et procede d'utilisation de cette derniere lors des analyses de sang
WO2004032750A1 (fr) * 2002-10-10 2004-04-22 Becton Dickinson And Company Systeme collecteur d'echantillons avec inhibiteur de caspase

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4132480A1 (de) * 1991-09-30 1993-04-08 Kabe Labortechnik Gmbh Vorrichtung zur blutentnahme
US5522804A (en) * 1994-02-15 1996-06-04 Lynn; Lawrence A. Aspiration, mixing, and injection syringe
DE19520883A1 (de) * 1995-06-08 1996-12-12 Szibor Reinhard Prof Dr Vorrichtung zur Blutentnahme mit einem zweikammrigen System
WO2000062061A1 (fr) * 1999-04-12 2000-10-19 Institut für Chemo- und Biosensorik Münster E.V. Dispositif et procede a utiliser lors de la realisation de tests relatifs a des systemes recepteur-ligand et de tests d'affinite
WO2002067778A2 (fr) * 2001-02-26 2002-09-06 Ben-Ami Ballin Seringue et procede d'utilisation de cette derniere lors des analyses de sang
WO2004032750A1 (fr) * 2002-10-10 2004-04-22 Becton Dickinson And Company Systeme collecteur d'echantillons avec inhibiteur de caspase

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107980764A (zh) * 2017-11-28 2018-05-04 上海原能细胞生物低温设备有限公司 一种低温中转箱
CN107980764B (zh) * 2017-11-28 2022-11-22 上海原能细胞生物低温设备有限公司 一种低温中转箱

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