EP2978838A1 - Procédé et dispositif servant à produire une culture de cellules à partir de cellules d'origine humaine ou animale - Google Patents

Procédé et dispositif servant à produire une culture de cellules à partir de cellules d'origine humaine ou animale

Info

Publication number
EP2978838A1
EP2978838A1 EP14713437.3A EP14713437A EP2978838A1 EP 2978838 A1 EP2978838 A1 EP 2978838A1 EP 14713437 A EP14713437 A EP 14713437A EP 2978838 A1 EP2978838 A1 EP 2978838A1
Authority
EP
European Patent Office
Prior art keywords
cells
cell culture
substrate
vessel
cell
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Withdrawn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
EP14713437.3A
Other languages
German (de)
English (en)
Inventor
Günter BERTHOLDT
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.)
Xellutec GmbH
Original Assignee
Xellutec 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 Xellutec GmbH filed Critical Xellutec GmbH
Publication of EP2978838A1 publication Critical patent/EP2978838A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • 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
    • 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
    • C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
    • C12M37/04—Seals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/78—Cellulose

Definitions

  • the invention relates to a method for producing a cell culture from human or animal cells according to the preamble of
  • Patent claim 1 a microtiter plate with a plurality of wells filled with a cell culture, according to the preamble of claim 12, and a use of a cell culture according to the preamble of patent claim 14.
  • human or animal mammalian cells The cell culture is exposed to the substances to be tested and the reaction of the cells is examined.
  • cell cultures are usually used today in which cells are grown on a surface such as a cell surface. cling to the bottom of a cell culture vessel. For this purpose, a suspension of human or animal cells is pipetted into a vessel in which a
  • Nutrient solution is included.
  • the cells then sediment in the nutrient solution and settle at the bottom of the vessel. There they attach and divide at a rate that is essentially dependent on the growth factors contained in the nutrient solution. After a few days, a so-called cell lawn forms on the bottom of the vessel, completely covering the soil. However, this cell lawn is stable only for a limited time. If the cells remain in the vessel too long - usually after a few days - the cells will lift off the bottom of the vessel and die. It is therefore necessary at regular intervals a so-called passage
  • the cells in which the cells from the bottom of the vessel by means of an enzyme detached, washed, isolated and then pipetted into another vessel for re-cell division. This process is called passenger and usually done by hand. He is relatively expensive and expensive.
  • the cells change after each passage, their phenotype and their specific properties. That is, they dedifferentiate and develop from a more differentiated stage with very specific properties, such as: As skin cells or liver cells, too nonspecific and ultimately for
  • the same medical test which is performed once on a cell culture of an older and once on a cell culture of a younger generation, can give different results. Therefore, the tests made on the known cell cultures are reproducible only for a very short period of a few days, as the cell culture changes over time.
  • Another disadvantage of cells that constantly divide is that the cells are in a condition that differs significantly from the situation in a tissue of an adult organism.
  • Cell division in an adult body is a relatively rare event. It usually occurs only after tissue loss and usually goes with one
  • a method for producing a cell culture from human or animal cells, in particular from mammalian cells, for carrying out medical or pharmacological tests comprises at least the following steps:
  • the cell is tested on the basis of metabolic parameters whether and when the cells have reached their characteristic state and - Supplying a drug to be tested to the on-substrate cell culture.
  • the cell culture is preferably maintained at about 37 ° C.
  • the cells cover the substrate so tightly that the outer membranes of the cells touch each other completely, the cells stop dividing.
  • the cells need a certain amount of time to change and reach a so-called dormant phenotype.
  • dormant refers only to the fact that there are no more dramatic changes in shape as in cell division.For biochemical reasons, these cells are very active and produce factors that are typical for their differentiation state microbially produced cellulose stable for at least several weeks.
  • the cells may be slow to storage or for transport
  • the cell culture is preferably prepared from stem cells or from cells obtained from stem cells, such. Liver, heart or kidney cells, or cells other organs, such as B. the skin.
  • stem cells such as Liver, heart or kidney cells, or cells other organs, such as B. the skin.
  • a medical or pharmacological test can thus be carried out on known, well-defined cells.
  • these cells may be e.g. Example, by means of a method, as it is known from US 20030161818A1 or US 2003 015 45 06 A1.
  • the stem cells are obtained from human umbilical tissue.
  • the stem cells are located in the so-called substantia gelatinea funiculi umbilicalis, a gelatinous matrix rich in hyaluronic acids and chondroitin sulfates, also called “wharton's jelly", which is extracted by an enzymatic process (eg collagenase).
  • for. B. cells are used, which come directly from affected patients, creating a
  • the cells applied to the cellulose substrate are preferably those cells which have not previously been passaged.
  • the cell culture thus arises according to the invention by a unique cell division and growth process of original, so-called.
  • Primary cells such.
  • stem cells or derived cells that have not gone through a dedifferentiation process That is, the cells applied to the cellulose substrate are preferably cells having a zero passage number.
  • the transport can rather be done at ambient temperature, such. B. between 15 ° C and 30 ° C.
  • the temperature can also be higher or lower.
  • the ambient temperature is within the desired range, e.g. B. between 15 ° C and 30 ° C, it is sufficient to allow the cell culture to cool easily (without using a cooling device).
  • the cell culture could also be active, ie by means of a cooling device to be cooled down to the desired temperature.
  • the vessel is provided with a hermetic seal, e.g. a foil or a lid provided.
  • the hermetic seal is intended to prevent the entry or exit of media such as liquid or gas from or into the vessel.
  • the sealing of the vessel can in principle be carried out before or after cooling, but is preferably before
  • the vessel is sealed with a foil, e.g. is welded or glued to the vessel.
  • a lid could be provided which closes off the vessel.
  • the vessel with the cell culture therein can finally be packaged, then z. B. to a laboratory or a customer, who then performs the medical or pharmacological tests on the cell culture.
  • the packaging may comprise, for example, a box and optionally insulating material.
  • the packaging may also include a coolant.
  • the nutrient solution can be mixed, for example, with methyl cellulose, polyethylene glycol or another viscosity-increasing substance.
  • a higher-viscosity substance such as B. a gel-like substance are introduced into the vessel. But it could also be a solid, such. B. a body of microcrystalline cellulose can be placed on the cell culture. As a result, the cell culture is mechanically held on the bottom of the vessel and can only be difficult to tilt to the side or float.
  • the cells of the cell culture are not passaged, ie the cell lawn is preferably not detached and the cells are not separated as in the prior art and then applied again to another substrate for re-cell division. Rather, the cell culture is preferably prepared by a unique cell division and growth process from original cells, ie, cells that have not previously been passaged.
  • the cell culture is preferably packaged in the same vessel in which it has been cultured and optionally sent to a recipient. It does not have to be transferred to another vessel.
  • the above method is performed on a microtiter plate having a plurality of wells.
  • a substrate of microbially produced cellulose is inserted, filled in a nutrient solution and applied to the substrate or human or animal cells. Thereafter, the cells grow and divide until at least partially covering the substrate.
  • each cell of the microtiter plate contains a part of the same cell culture. The individual subcultures can then be used to perform the same or different medical or pharmacological tests.
  • the storage or transport of the cell culture is preferably carried out at
  • the entire microtiter plate is preferably sealed with a hermetic
  • the invention also relates to a microtiter plate having a plurality of wells, each filled with a substrate made of microbial cellulose which is populated by a cell culture.
  • the microtiter plate is located
  • a substance which is arranged on the cell culture and has a higher viscosity than the nutrient solution is contained in the individual wells of the microtiter plate.
  • the cell culture is held together with the substrate mechanically at the bottom of the vessel and can only be difficult to tilt to the side or float.
  • the invention also relates to a use of a cell culture from human or animal cells, in particular mammalian cells, which has been prepared according to one of the methods described above, for carrying out medical or pharmacological tests.
  • Fig. 1 a - 1 h different states of a method for producing a
  • Fig. 2 is a microtiter plate with several wells in which
  • Figures 1 a-1 h show various states of a method for
  • the cell culture 9 can later be used to perform medical or pharmacological tests.
  • Nutrient solution 2 filled (Fig. 1 a).
  • nutrient solution 2 for example, the nutrient medium of Schramm and Hestrin described in Biochemical Journal 58 of 1954, pages 345-352 can be used.
  • the nutrient solution may, for example, consist of 20 g of glucose, 5 g of yeast extract, 5 g of bactopeptone, 2.7 g of sodium phosphate and 1.15 g Citric acid monohydrate and 0.5 g of magnesium sulfate heptahydrate in one liter of water.
  • other nutrient solutions known in the art may also be used.
  • a substrate 3 made of microbial cellulose which may for example have the form of a platelet, is then placed in the vessel 1 (FIG. 1 b).
  • the two steps described above can also be performed in reverse order.
  • a substrate 3 of microcrystalline cellulose can be produced, for example, by means of a method as known from DE 10 2008 056 413.3 or WO 2010 052 019 A2.
  • microcrystalline cellulose are well known in the art.
  • a suspension of human or animal cells 4 is finally introduced into the vessel 1.
  • the applied to the substrate 3 cells can, for. B. stem cells or cells derived from stem cells, such as. As liver, heart or kidney cells, or cells of other organs, such as. B. the skin.
  • the cells 4 can z.
  • Example be prepared by a method, as it is known from US 02013025983A1, US 6410320B1 or US 7534607B1.
  • the cells 4 applied to the cellulose substrate are preferably those cells which have not previously been passaged.
  • the cell culture 9 thus arises from a unique cell division and growth process of original cells that have not undergone a dedifferentiation process. That is, the cells 4 applied to the cellulose substrate 3 are preferably cells having a passage number equal to zero.
  • the cells 4 then sediment in the nutrient solution 2 and settle on the surface of the cellulose substrate 3. At temperatures around 37 ° C then takes place a division and growth process, which forms on the substrate 3 within a few days, a so-called cell lawn 5, which completely covers the substrate 3, as shown in Fig. 1 d. Once the cell lawn 5 has reached the desired size and thickness, the cell division and growth process stops.
  • the vessel 1, including its contents, is then cooled to a lower temperature. According to a preferred embodiment of the invention, the vessel is allowed to cool to room temperature, e.g. B. to about 18 ° C to 25 ° C. It has been found that the cell culture 9 at these temperatures over several weeks, z. B.
  • an agent 10 increasing the viscosity of the nutrient solution 2 such as e.g. Methyl cellulose or polyethylene glycol (PEG) are added. This results after the addition of the agent 10
  • Cellulose can be placed on the cell culture 9. This can be prevented that the substrate 3 lifts off together with the cell culture 9 from the bottom of the vessel 1 and z. B. tilts or floats to the side. In a further step 1f, the vessel 1 can still be hermetically sealed
  • the hermetic seal 6 may be, for example, a foil which may be e.g. can be welded or glued to the vessel 1.
  • a lid with a seal could be provided.
  • the vessel 1 is finally packed in step 1 g.
  • the vessel 1 is packed in a box 12 and additionally protected by a filler 13 against mechanical shocks and damage.
  • the cell culture 9 is preferably shipped in the same vessel 1 in which it has also been cultivated.
  • FIG. 1 h also shows how a substance 14 to be tested is applied to the cell culture 9, while the cell culture 9 is still located on the substrate 3. The reaction of the cells to the substance is then diagnosed.
  • FIG. 2 shows a microtiter plate 8 with a plurality of wells 7, each serving as a vessel 1 according to FIGS. 1 a to 1 g.
  • a substrate 3 of microcrystalline cellulose which is populated by a cell culture 9.
  • the preparation of the cell cultures 9 can be produced simultaneously in accordance with the method steps shown in FIGS. 1a to 1d.
  • the microtiter plate 8 is cooled to room temperature and z. B. closed by means of a film 6, as shown in Fig. 1f. In each of the wells 7 is then part of the same cell culture.
  • microtiter plate 8 If the microtiter plate 8 is to be shipped, it is packed in a further step according to FIG. 1 g. The customer thus receives one

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Molecular Biology (AREA)
  • Clinical Laboratory Science (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé servant à produire une culture de cellules (9) à partir de cellules d'origine humaine ou animale, en particulier de cellules issues de mammifères, afin de mettre en œuvre des essais médicaux ou pharmacologiques. Le procédé selon l'invention comprend au moins les étapes suivantes consistant à : placer un substrat (3) composé de cellulose préparée par voie microbienne dans un récipient (1) ; remplir le récipient (1) d'une solution d'éléments nutritifs (2) ; appliquer sur le substrat (3) des cellules (4) d'origine humaine ou animale ; réguler la température du contenu du récipient de manière à le porter à la température corporelle ; et attendre que le substrat (3) soit complètement recouvert par un tapis de cellules (9), en d'autres termes jusqu'à l'obtention d'une confluence. Il est alors possible de mettre en œuvre sur la culture de cellules (9) ainsi produite des essais médicaux ou pharmacologiques.
EP14713437.3A 2013-03-25 2014-03-25 Procédé et dispositif servant à produire une culture de cellules à partir de cellules d'origine humaine ou animale Withdrawn EP2978838A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102013005198.3A DE102013005198B4 (de) 2013-03-25 2013-03-25 Verfahren und Vorrichtung zum Herstellen einer Zellkultur aus menschlichen oder tierischen Zellen
PCT/EP2014/055940 WO2014154679A1 (fr) 2013-03-25 2014-03-25 Procédé et dispositif servant à produire une culture de cellules à partir de cellules d'origine humaine ou animale

Publications (1)

Publication Number Publication Date
EP2978838A1 true EP2978838A1 (fr) 2016-02-03

Family

ID=50390080

Family Applications (1)

Application Number Title Priority Date Filing Date
EP14713437.3A Withdrawn EP2978838A1 (fr) 2013-03-25 2014-03-25 Procédé et dispositif servant à produire une culture de cellules à partir de cellules d'origine humaine ou animale

Country Status (5)

Country Link
US (1) US20160024462A1 (fr)
EP (1) EP2978838A1 (fr)
CN (1) CN105051182A (fr)
DE (1) DE102013005198B4 (fr)
WO (1) WO2014154679A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108300713A (zh) * 2017-12-31 2018-07-20 宁波大学 固定细胞的方法及装置

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6410320B1 (en) 1992-03-02 2002-06-25 The University Of Michigan Method and compositions for isolation and growth of kidney tubule stem cells, in vitro kidney tubulogenesis and ex vivo construction of renal tubules
US20030154506A1 (en) 2002-01-29 2003-08-14 Gin Wu Process of generating stem cells equivalent to human embryonic stem cells
US20030161818A1 (en) 2002-02-25 2003-08-28 Kansas State University Research Foundation Cultures, products and methods using stem cells
WO2006042287A2 (fr) * 2004-10-12 2006-04-20 Trustees Of Tufts College Procede de production d'echafaudages de biomateriaux
US7534607B1 (en) 2005-12-27 2009-05-19 Industrial Technology Research Institute Method of producing cardiomyocytes from mesenchymal stem cells
DE102008056413B4 (de) * 2008-11-07 2014-12-24 Bioregeneration Gmbh Verfahren zur Herstellung eines Cellulose enthaltenden Körpers
DE102010006207B4 (de) 2010-01-29 2022-06-23 Zf Active Safety Gmbh Scheibenbremse mit reduziertem Restschleifmoment

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2014154679A1 *

Also Published As

Publication number Publication date
DE102013005198A1 (de) 2014-09-25
DE102013005198B4 (de) 2016-05-19
US20160024462A1 (en) 2016-01-28
CN105051182A (zh) 2015-11-11
WO2014154679A1 (fr) 2014-10-02

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