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WO2016128171A1 - Plaque de culture cellulaire - Google Patents

Plaque de culture cellulaire Download PDF

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Publication number
WO2016128171A1
WO2016128171A1 PCT/EP2016/050746 EP2016050746W WO2016128171A1 WO 2016128171 A1 WO2016128171 A1 WO 2016128171A1 EP 2016050746 W EP2016050746 W EP 2016050746W WO 2016128171 A1 WO2016128171 A1 WO 2016128171A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell culture
pedestal
culture plate
insert
liquid
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.)
Ceased
Application number
PCT/EP2016/050746
Other languages
German (de)
English (en)
Inventor
Moriz Walter
Andreas Traube
Tobias Brode
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.)
Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
Original Assignee
Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
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 Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV filed Critical Fraunhofer Gesellschaft zur Foerderung der Angewandten Forschung eV
Publication of WO2016128171A1 publication Critical patent/WO2016128171A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C12M25/04—Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates

Definitions

  • the invention relates to improved cell culture plates for the in vitro cultivation of biological cells and tissue as well as improved cultivation methods using such cell culture plates.
  • Cell culture plates also referred to as microtiter plates or multiwell plates, are known for the cultivation of biological cells or tissues both in the laboratory and especially in automated production of artificial biological tissue ("tissue engineering"), which have an outer, generally rectangular, surface and in the dimensions of standardized frames and usually contain several regularly arranged wells, so-called “wells", for receiving cell culture medium in which biological cells or tissue pieces can be incubated and cultivated in parallel approaches to each other.
  • tissue engineering artificial biological tissue
  • a particular variant of this is a cell culture plate with cup-shaped cell culture inserts which can be used therein, the so-called “inserts.”
  • These cell culture inserts generally have a semipermeable membrane or a net on the bottom side for accommodating biological cells or tissue
  • the biological cells or tissue cultivated therein are then in direct contact with cell culture medium both on the top side and on the bottom side, and can be supplied with nutrients, among other things, with cell culture medium within the respective cup-shaped cell culture insert and with cell culture turmediunn in the recess of the cell culture plate on the other hand.
  • these cell culture inserts, together with the biological cells or tissues cultured therein can be easily removed from the cell culture plate and otherwise processed further.
  • Cell culture plates for receiving such cell culture inserts thus have clear advantages in the cultivation of cells and tissue compared to such multiwell plates, wherein the cells or tissues are cultivated directly on the bottom of the respective well.
  • known arrangements of cell culture plate and cell culture inserts still have disadvantages in practical application: it has especially in the handling of a cell culture plate filled with cell culture medium and / or a necessary change or replacement of cell culture medium proved to be particularly disadvantageous that the cells and tissue cultivated in the cell culture inserts are mechanically impaired by occurring local currents and turbulence of the medium or even fall dry at times.
  • the problem here is that, especially at the cell culture inserts, especially in the region of the bottom-side membrane, in particular because of their porous structure, air bubbles form and remain trapped there even after the change of media. This leads to a local media and thus nutrient undersupply of the biological cells and tissue, which can lead to the death of the entire piece of tissue as the culturing period progresses.
  • high purge rates must be used to eliminate the dropped air bubbles and trapped air due to purge pressure.
  • this is, as stated above, mechanically very stressful for the cultured cells or tissues.
  • the invention was based on the technical problem of improving known cell culture plates for receiving cell culture inserts in such a way that the abovementioned disadvantages can be eliminated and improved, and also alternative cultivation conditions can be created for cells and tissues cultured therein.
  • the technical problem is completely solved by the provision of a novel designed cell culture plate, suitable for the inclusion of known cell culture inserts for the cultivation of biological cells or tissues, having the features of claim 1
  • the invention in this first aspect is a cell culture plate having at least one trough-shaped depression (well) for receiving cell culture inserts (inserts) and liquid cell culture medium.
  • the at least one recess has one or more pedestals which can each be assigned to the cell culture inserts which can be accommodated in the recess.
  • one (1) usually cup-shaped cell culture insert element has exactly one (1 ) Podium assigned to the pit.
  • one (1) cell culture insert in the cell culture plate is associated with a plurality of pedestals of the well, but preferably arranged in a group.
  • a plurality of cell culture inserts, preferably arranged in a group, in the cell culture plate are associated with precisely one (1) pedestal of the well.
  • the pedestal and the receptive cell culture insert are now spaced apart from one another such that a local distance space can form between the top side of the pedestal and the bottom side of the cell culture insert when the cell culture insert is inserted there into the cell culture plate.
  • the distance, and thus the size of the local distance space is predeterminable or adjustable by the structural dimensioning of the cell culture plate and the pedestal.
  • the invention provides in particular that the local distance space formed by the spacing of pedestal and ingestible cell culture insert can accommodate liquid cell culture medium due to the known co-operation of cohesive and adhesive forces, according to the invention independent of the actual level of the cell culture medium in the well itself.
  • the stationary fluid meniscus formed there from culture medium can effectively suppress or prevent a mechanical impairment of the cells or tissue and also the disadvantageous dry-trapping. It turns out that this distance space represents a flow obstacle, as a result of which a local "calming zone" is formed and effective turbulences of the cell culture medium in the area of the membrane of the ingestible cell culture insert can be effectively reduced or completely suppressed
  • the volume of the liquid meniscus which can be formed in the local distance space can be determined by selecting the spacing of the pedestal and the receptive cell culture insert
  • the fluid meniscus that can be formed in a local distance space preferably has a volume of 100 to 350 ⁇ , more preferably from 150 to 250 ⁇ .
  • the liquid meniscus which can be generated according to the invention in the local distance space is advantageously a residual volume which, in the event of a required media change, results from the depression of the cell space. culture plate on the membrane of the cell culture insert, so as to "buffer off" mechanical, physical and / or chemical disturbances on the tissue, which would otherwise be accompanied by a medium change.
  • the liquid meniscus according to the invention permits small volumes below the same in the same way to work, for example, dyeings and drug tests efficiently and quickly, since only a small volume, preferably up to 350 ⁇ , must be used or exchanged below each cell culture insert the small liquid volume below the cell culture insert also allows novel tests with high temporal resolution, since this small volume of liquid is quickly exchangeable.
  • the cell culture plate thus permits the bottom-side application of cell culture medium only in the region of the distance space formed, with the remainder of the well of the cell culture plate, in which the cell culture insert is suspended, remaining unfilled.
  • This is particularly interesting in connection with the application of solid substances and active substances to the biological cells and tissue from the underside of the cell culture insert, since due to the small volume of the distance space, a rapid change of media, for example by pipetting, can take place and / or simultaneously only one low culture media must be used.
  • staining with the so-called MTT essay can be carried out particularly simply and efficiently with the cell culture plate according to the invention, with a particularly low volume of the MTT reagent can be given under the cell culture insert.
  • this can preferably be restricted to a volume of about 100 to 350 .mu.l, whereas in current cell culture plates, in some cases several milliliters of a reagent must be added to the entire well in order to adequately wet the cells and tissue in the cell culture inserts.
  • Cell culture inserts which can be used according to the invention are known per se, and have on the bottom side a cell culture membrane or network, whereupon the biological cells or tissue rest during the incubation and cultivation and can optionally adhere. Multiple cell culture inserts may be grouped together in a frame. Inserts for cell culture plates thus consist in particular of a frame and a plurality of cup-shaped cell culture inserts arranged in series; Frame and cup-shaped cell culture inserts are particularly integrally formed, for example as an injection molded part, with cell culture membranes used therein. The inclusion of the cell culture insert in the cell culture plate is carried out individually or in particular in the form of a plurality of rows of cell culture inserts arranged in rows in a common frame.
  • the cell culture or the inserts are placed in a first embodiment in the cell culture plate or in particular mounted on a formed on the cell culture insert frame member.
  • it is provided to clamp the cell culture insert or the individual cells, individually or in a frame, into the cell culture plate and to hold it in a form-fitting and preferably frictionally engaged manner.
  • the cell culture insert itself or its frame can have, in particular, an oblique chamfer or a conical shape which engages with a counter-structure of the cell culture plate.
  • the top of the pedestal is adapted in shape to the shape of the underside, especially the culture membrane, of the respective cell culture insert.
  • the pedestal therefore preferably has essentially the shape of a cylinder or conical stump with a preferably circular cut surface which forms its top side.
  • the top of the pedestal is preferably oriented parallel to the bottom of the cell culture plate.
  • the pedestal essentially has the shape of a cuboid, cube or a truncated pyramid with a preferably rectangular or square cut surface.
  • the cell culture plate according to the invention thus allows a strong reduction of the process times by a faster exchange of culture medium or of test reagents.
  • the cell culture plate according to the invention also allows cell culture to be carried out in one and the same cell culture plate using the total volume of the cell culture plate and subsequently or in an intermediate step to reduce the liquid volume to the fluid meniscus, there by means of test reagents and similar tests Perform live staining without having to change the cell culture plate or need to be replaced.
  • the pedestal on the top side at least one spacer (spacer) for determining / fixing the distance of the cell culture insert can be accommodated there from the pedestal.
  • the spacer or spacers are preferably on the circumference of the pedestal.
  • at least two opposing spacers are provided on the pedestal, more preferably four opposing spacers.
  • six or more spacers are arranged in a ring shape on the circumference of the pedestal. It is provided that between the spacers only a narrow channel remains, what the volume within the distance space communicates with the volume outside the distance space, that is, with the remaining volume of the recess. By arranging the spacers and the spacing between them, the degree of liquid exchange and thus the flow obstacle between the liquid meniscus formed in the distance space and the surrounding volume in the depression can be adjusted. At the same time or as an alternative, the volume of the fluid meniscus in the distance space, in particular in conjunction with the surface properties of the pedestal and spacer, the capillary properties and the interaction of cohesive and adhesive forces, can thus also be determined.
  • a spacer for determining the Abstands may serve, in particular in conjunction with another, at least opposite spacers, in addition to the fixation and / or positioning of the recordable cell culture insert.
  • the spacer as such in a corresponding structure in the cell culture insert as it were “lock” form-fitting and thus determine the position.
  • a groove may be provided in the spacer, wherein a corresponding counter-structure may engage the cell culture insert.
  • the insertable cell culture insert preferably can be held in a form-fitting manner at its lower circumferential edge.
  • the invention further provides that, depending on the field of application, technical problem or stage of cultivation, one and the same cell culture insert in which the biological cells or tissue are just cultured, in each case different cell culture plates according to the invention are used, in particular by the spacing of the pedestals to the cell culture inserts - and thus in the size of the local distance space and there formed fluid meniscus - different.
  • the invention also provides a cell culture plate, wherein the at least one pedestal has at least one lateral extension at least on the upper side, which projects beyond the cross section of the pedestal hidden by the ingestible cell culture insert and thus allows direct access from above to the distance space under the cell culture insert.
  • the lateral extension thus offers access, for example via a pipetting robot, from above, laterally past the inserted cell culture insert, to the liquid meniscus in the local distance space under the cell culture insert.
  • the extension may be formed at a single location on the pedestal, but may also be formed at several locations on the perimeter of the pedestal. Alternatively, this extension can also be designed as a peripheral annular zone of the pedestal, outside the area of the upper side of the pedestal covered by the cross section of the inserted cell culture insert. In this case, the diameter of the pedestal is simply greater than the diameter of the cell culture insert used there.
  • the at least one extension is located at the point at which recesses for the access of pipette tips to the bottom of the recess of a standard culture plate are provided in conventional cell culture plates and cell culture inserts. In a preferred embodiment, this extension can expand and set the volume of the liquid miniscus formed in the spacer space by appropriate dimensioning.
  • the invention also takes advantage of the interaction of adhesion and cohesion of the liquid on the surfaces of the cell culture plate and the cell culture insert.
  • the volume of the fluid meniscus beneath the cell culture insert can be predetermined in addition or alternatively to the choice of the distance of the cell culture application from the pedestal.
  • the invention also contemplates, alternatively or additionally, determining the volume of the fluid meniscus beneath each cell culture insert by adjusting the hydrophobicity and thus the wetting angle of the respective surfaces, particularly the surface of the pedestal. By changing the wetting angle, the volume of the liquid meniscus can be adjusted. It is understood that the cell culture plate is essentially suitable and intended for use with aqueous media. A transfer of the conditions to the use of oily media and the resulting surface properties can now readily be adapted by those skilled in the art, having regard to the teaching described herein.
  • the cell culture plate in the side wall forming the trough-shaped depression has at least one separate access, which preferably extends to the bottom of the depression. Via this access, the at least one depression of the cell culture plate can be emptied or filled from the outside without otherwise opening the cell culture plate must become. For example, a lid applied to protect the culture on the cell culture plate may remain.
  • the invention provides in particular that the at least one access is arranged at least or exclusively on the short end face of the usually rectangular cell culture plate. By positioning the access on the short side, the plate can continue to be handled with commercially available gripping systems, particularly in automated processes.
  • the invention also relates to a method for culturing biological cells or tissues in vitro in or on a cell culture insert, which is used in a well of a cell culture plate according to the invention, wherein the method is characterized in that liquid cell culture medium during the cultivation of biological cells or Tissue is maintained as a level-independent liquid meniscus between the cell culture insert and the pedestal of the cell culture plate.
  • liquid cell culture medium is kept in the form of a fluid meniscus only in the distance space between cell culture insert and pedestal; more preferably, no cell culture medium is located at the bottom of the well of the cell culture insert outside the pedestal.
  • the method according to the invention for culturing biological cells or tissues in vitro in the cell culture plate according to the invention comprises at least one process of media change of liquid cell culture medium, whereby old cell culture medium present in a first step of the medium change is removed from the bottom of the well and transferred to the cell culture plate a fresh cell culture medium is introduced into the depression in a subsequent step, whereby in both steps, that is also after the removal of far from the Zellkulturnnediunns from the bottom of the well and before filling the fresh cell culture medium always a liquid meniscus of the cell culture medium between cell culture insert and pedestal of the cell culture plate remains.
  • the invention also provides for the use of a base-side pedestal in a depression of a cell culture plate according to the invention for the purpose of forming a liquid meniscus independent of the media fill level of the well under a cell culture insert insertable into the depression.
  • the volume of the liquid meniscus which can be formed in the local distance space can be predetermined by the choice of the height of the spacer on the pedestal to the receptive cell culture insert.
  • Figure 1 shows an oblique view of a first embodiment of the cell culture plate 10 in a special form with a single trough-shaped recess 12 for receiving Zellkultureins2011- zen. From the bottom of the recess 12 regularly arranged pedestals 30 protrude upwards. At each pedestal 30 four pairs of opposing spacers 32 are formed here. In addition, the pedestals 30 each have a lateral extension 38 at least on top. In addition, the cell culture plate 10 has an access 16 to the trough-shaped depression 12.
  • FIG. 2 shows a sectional view of the cell culture plate according to FIG. 1, in which cell culture inserts 20 are inserted.
  • the podiums 30 are in or formed on the bottom 14 of the recess 12.
  • the bottom side 22 of the cell culture insert is fixedly spaced from the pedestals 30 in the inserted state via the spacers 32 formed on the pedestals 30, so that a spacer space 36 is formed between the bottom 22 of the cell culture insert 20 and the pedestal 30 rising from the recess 12 contains a level-independent liquid meniscus in the ready state.
  • a shoulder 34 is provided on each of the spacers 32 and engages the bottom 22 of the cell culture insert 20 to secure the cell culture insert 20 at least in a horizontal direction (lateral) in position. Not shown in this sectional drawing are formed on the pedestals 30 side extensions 38th
  • FIG. 3 shows an oblique and sectional view of the cell culture plate 10 according to FIGS. 1 and 2.
  • FIG. 4 shows a top view of the cell culture plate 10 according to FIGS. 1, 2 and 3.
  • Figure 5 shows schematically in plan view various embodiments of the arrangement of the spacers 32 on the pedestal 30.
  • the connection provides at least two opposing spacers 32, which are preferably each provided with a shoulder 34 (Figure 5a).
  • a plurality of spacers are arranged along the circumference of the pedestal along a crown, wherein the intermediate space formed between the spacers are dimensioned such that thus the liquid exchange between the distance space and the environment is set (Figure 5c).

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  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Clinical Laboratory Science (AREA)
  • Immunology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne une plaque de culture cellulaire (10) présentant au moins un renfoncement en cuvette (well = puits) (12) destiné à recevoir des inserts de culture cellulaire (inserts) (20) et un milieu fluide. Ladite plaque de culture cellulaire se caractérise en ce que le renfoncement (12) présente au moins un socle (30) pouvant être associé aux inserts de culture cellulaire (20) pouvant être reçus, de sorte qu'un écart pouvant être prédéfini entre le socle (30) et un insert de culture cellulaire (20) qui y est introduit permet de former un espace intermédiaire (36) local entre le socle (30) et l'insert de culture cellulaire (20).
PCT/EP2016/050746 2015-02-11 2016-01-15 Plaque de culture cellulaire Ceased WO2016128171A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102015202402.4A DE102015202402B3 (de) 2015-02-11 2015-02-11 Zellkulturplatte
DE102015202402.4 2015-02-11

Publications (1)

Publication Number Publication Date
WO2016128171A1 true WO2016128171A1 (fr) 2016-08-18

Family

ID=53782770

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2016/050746 Ceased WO2016128171A1 (fr) 2015-02-11 2016-01-15 Plaque de culture cellulaire

Country Status (2)

Country Link
DE (1) DE102015202402B3 (fr)
WO (1) WO2016128171A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114874901A (zh) * 2022-05-16 2022-08-09 广州市华粤行医疗科技有限公司 一种介质承载容器及介质的培养方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5534227A (en) * 1995-06-05 1996-07-09 Becton, Dickinson And Company Thermoform dish insert
WO2004020571A2 (fr) * 2002-08-30 2004-03-11 Oxyphen Ag Insert de culture de cellules
DE202006009787U1 (de) * 2006-06-22 2006-08-24 Wilden Ag Anordnung zur Schaffung von räumlichen Strukturen aus organischen Zellen
WO2006131123A2 (fr) * 2005-06-10 2006-12-14 Nunc A/S Support d'element d'insertion de culture, element d'insertion de culture et systeme d'elements d'insertion de culture
WO2011127945A1 (fr) * 2010-04-15 2011-10-20 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Système de culture cellulaire

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10321042B4 (de) * 2003-01-17 2006-09-21 Greiner Bio-One Gmbh Biochip-Träger
JP4586192B2 (ja) * 2005-03-08 2010-11-24 財団法人生産技術研究奨励会 細胞培養チャンバー

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5534227A (en) * 1995-06-05 1996-07-09 Becton, Dickinson And Company Thermoform dish insert
WO2004020571A2 (fr) * 2002-08-30 2004-03-11 Oxyphen Ag Insert de culture de cellules
WO2006131123A2 (fr) * 2005-06-10 2006-12-14 Nunc A/S Support d'element d'insertion de culture, element d'insertion de culture et systeme d'elements d'insertion de culture
DE202006009787U1 (de) * 2006-06-22 2006-08-24 Wilden Ag Anordnung zur Schaffung von räumlichen Strukturen aus organischen Zellen
WO2011127945A1 (fr) * 2010-04-15 2011-10-20 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Système de culture cellulaire

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114874901A (zh) * 2022-05-16 2022-08-09 广州市华粤行医疗科技有限公司 一种介质承载容器及介质的培养方法

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