[go: up one dir, main page]

WO2021048441A1 - Procédé et appareil pour l'isolement de cellules et cellules isolées pour la cicatrisation - Google Patents

Procédé et appareil pour l'isolement de cellules et cellules isolées pour la cicatrisation Download PDF

Info

Publication number
WO2021048441A1
WO2021048441A1 PCT/EP2020/075653 EP2020075653W WO2021048441A1 WO 2021048441 A1 WO2021048441 A1 WO 2021048441A1 EP 2020075653 W EP2020075653 W EP 2020075653W WO 2021048441 A1 WO2021048441 A1 WO 2021048441A1
Authority
WO
WIPO (PCT)
Prior art keywords
filter
enzyme
solution
pieces
cells
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/EP2020/075653
Other languages
English (en)
Inventor
Christopher DUNNIL
Nikolaos GEORGOPOULOS
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.)
Veritacell
Original Assignee
Veritacell
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 Veritacell filed Critical Veritacell
Priority to EP20788982.5A priority Critical patent/EP4028035A1/fr
Publication of WO2021048441A1 publication Critical patent/WO2021048441A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the present invention relates to a method and method of use of apparatus to obtain and provide the cells for treatments such as re-epithelisation, decreased scaring and pigmentation reduction.
  • surgeons would harvest the dermal-epidermal portion’ of the skin layer from a donor area of a patient using a specific knife fit for the purpose and then apply the harvested skin layer to the wound.
  • the properties of the skin graft provide an environment for the treated wound to start to heal.
  • the donor site is then managed to heal using standard dressings.
  • This skin graft approach typically is used to treat very large wounds or burns and wounds that are struggling to heal.
  • the current art requires the patient to have a graft harvested, the graft taken away processed in a special heating device, carefully rinsed to remove the animal materials to prevent a non-host reaction, prepared in the delivery devices and then applied to the wound. Due to the time involved in processing the graft this can often be two or more procedures, or is a long period with the patient under sedation or anaesthesia. Both of these issues are clinically significant in the clinicians’ mind and inducing a trade-off decision between the risk of the procedure and the benefit of the treatment.
  • a skin graft is taken, usually a split-thickness skin graft (STSG) is taken (epidermis and very thin section of dermis) using a range of devices such as manual and electric dermatomes or a Humby knife and this may vary between surgeons.
  • STSG split-thickness skin graft
  • the graft can then be cut before exposure to enzymes.
  • RenovaCare® centrifuge the cells into a pellet, aspirate the solution, then re-suspend the pellet.
  • Another method used by Avita® employs trypsin (porcine) for 15-45 minutes at 37°C, they use an electric system integrated into the kit (integrated heating element) that is operated by AA batteries to heat the enzyme solution to this temperature. They reduce enzyme activity by dilution of the enzymatic solution.
  • RenovaCare® isolates cells from digested unwanted tissue (mainly dermis) mixed together in solution by passing it through a 40 pm pore size strainer and then again by centrifuging them and re-suspending the pellet in buffer (as mentioned above).
  • the Avita® system passes digested unwanted tissue containing solution through a 100 pm pore size strainer only and this completes their isolation of cells.
  • RenovaCare® use a technologically advanced electric spray gun to disperse the suspension on the wound and Avita® uses a relatively simple spray cap and tube.
  • a method of isolating cells including the step of taking a skin graft from a patient and characterised in that said graft is then exposed to human (non-animal) recombinant trypsin.
  • the trypsin does not contain, or is void of, other proteases.
  • the graft is exposed to the trypsin enzyme for 20-30 minutes. Typically the exposure is at room or ambient temperature. Further typically the temperature is approximately 22°C (operating theatre temperature).
  • the graft tissue is cut into approximately 1-2 mm 2 pieces.
  • the graft tissue is cut into substantially 1 mm 2 pieces.
  • a relatively long blade 5-7cm
  • an anti-blunting surface similar to plastic (or multiple-ply foam) chopping board material is used as a surface to cut the tissue.
  • excess enzyme fluid is then drained. Typically the fluid is drained after approximately 20-mins. Further typically the fluid is drained using negative or positive pressure. This leaves mainly and only enzyme that has soaked into the tissue. This is to avoid losing cell into the concentrated enzyme mixture which would eventually lead to cell damage.
  • the skin pieces are monitored for dermal-epidermal separation.
  • the tissue pieces are either agitated in-situ, typically using the scalpel blade or syringe tip for 2-5 minutes.
  • turbulent liquid mixing using the syringe is performed to release cells and heavily dilute the enzyme.
  • the solution may be placed on ice in the collecting tube to strongly deactivate the already diluted enzyme.
  • the material is minced or diced before straining.
  • mincing uses an fop and down’ directional cutting method similar to dicing.
  • Preferably excess enzyme or enzyme solution is drained first.
  • a filter or strainer is used to remove unwanted tissue.
  • a single filter is used.
  • the filter is a 4-10 pm filter. Further preferably the filter is a 5 pm filter.
  • a first or preliminary filter or strainer of larger size or larger mesh pore diameter is used.
  • a 40-100 pm strainer is used as a first or preliminary filter.
  • a 50-90 pm strainer is used.
  • a 70 pm first filter is used first, although the invention has been found to work well with a single small filter of pore diameter 4-10 pm.
  • a second filter is employed a 5 pm filter is used.
  • the second filter is a 4-10 pm filter.
  • recovered or isolated cells are placed into a buffered solution.
  • the isolated cell solution is applied to a wound.
  • the solution is dripped and/ or flowed onto the wound. Further typically the solution is applied to the wound by syringe.
  • a method for isolating cells including the steps of taking a skin graft and exposing the same to at least one enzyme and characterised in that after enzyme treatment a single filter or strainer is used to remove at least some unwanted tissue.
  • the filter is a 5 pm filter.
  • the filter is a 4-10 pm filter.
  • the one or more enzymes include human trypsin.
  • a pressure gradient is applied across the filter to push and/or draw the material through the filter.
  • a negative pressure is applied to draw the filtrate through the strainer.
  • the filter removes dermal fibres (long strings of extra-cellular matrix proteins) behind which provides a better yield and a uniform Ton-aggregated’ cell suspension.
  • a novel, more efficient, more convenient, more consistent and easier- to-use way of isolating cells so they can be applied to a wound and improve the healing of said wound said method including the steps of: taking a skin graft or section, cutting the graft into pieces, placing the pieces in an enzyme solution, incubating the combination of pieces and enzyme solution for a period of time, rinsing the resultant materials and solution through a strainer or filter, collecting the filtered solution and applying the cells from the filtered solution and/ or filtered solution to a wound.
  • the cutting of the skin graft or section into pieces involves pieces being created that are between 1 mm 2 in surface area and 2 mm 2 in surface area. Preferably between 1 mm 2 and 2 mm 2 and more preferably 1 mm 2 .
  • the skin pieces are placed in an enzyme solution containing a tissue digestion enzyme.
  • a tissue digestion enzyme Preferably the enzyme is trypsin. More preferably the enzyme is human sourced trypsin.
  • the enzyme concentration should be between 0.1-3%.
  • the mix of cells and enzyme are incubated together between 30 seconds and 36 hours at a temperature between -5 °C and 55 °C.
  • the temperature range should be 4 °C and 39 °C.
  • the incubation time is between 10 minutes and 6 hours. More preferably the temperature is between 4 °C and room temperature.
  • the incubation time is between 15 minutes and 1 hour.
  • the skin graft and enzyme solution are incubated or mixed together in a vessel in a room without need for further equipment in the room.
  • this mixture is held in a shaped device that allows the pieces and enzyme to be in solution.
  • the mixture is held in a device that allows easy access of graft pieces and enzyme solutions, that allows the device to be placed in a mechanical agitator or persons hand, and said device held easily by clinical staff for the duration of incubation, and said device can be emptied easily.
  • the incubated or digested material is emptied from the device onto a porous material, that allows the target skin cells or target skin cell containing solution to be separated from the digested tissue components.
  • a porous material that allows the target skin cells or target skin cell containing solution to be separated from the digested tissue components.
  • this material includes a filter or porous membrane.
  • the filter or porous membrane comprise Polyethylene terephthalate.
  • the material is agitated to speed the separation of the cells.
  • the separation material is part of the device used in the incubation process, and or connected to the device that the cells are collected in or the waste material is collected in. In one embodiment the separated cells are collected in a device for storage or for application to the wound.
  • the device is the device that is used to deliver the cells to the wound. More preferably the device facilitates the directional application of cells to the wound. Further preferably the device allows the controlled placement of cell solution at one or more sites within and/ or on the wound. Typically all or part of the equipment used in the process is all disposable within the normal waste disposal processes within the treatment area. Preferably the equipment can all be disposed of within the clinical waste stream. Typically during the conduct of the whole process, from harvest to cell application, no further equipment in addition to that normally found in a clinical setting is needed. In one embodiment the procedure for treating the graft and harvesting the cells requires no more equipment than is found than in the equipment supplied as part of the invention.
  • an apparatus for recovering or isolating cells said apparatus includes one or more filters or strainers.
  • the apparatus is supplied as a kit or packaged as a kit of parts.
  • the apparatus includes any one or any combination of the following; sharp chopping blade and/or surgical scalpel, sterile rubber-like surface (anti-blunting), three ply foam mat, pluristrain ring connector (allows negative pressure), small trench next to the chopping surface to capture tissue in, small well or tube that fits the strainers, enzyme solution, buffer solution, first and second strainers, 5 or 10 ml sterile syringe.
  • Figure 1 illustrates tissue mincing procedure for cell suspension preparation on appropriate plastic surface
  • Figure 2 illustrates evidence of aggregation of epithelial cells on dermal fibres (extracellular matrix proteins) in the absence of straining
  • Figure 3 illustrates use of negative pressure and straining to isolate the viable epithelial cells; and Figure 4 illustrates evidence of lack of any aggregation of epithelial cells following straining due to the absence of dermal fibres (extracellular matrix proteins).
  • the present invention has been described predominantly for harvesting skin cells to apply to skin wounds. It is within the art that other tissues may be used and cells extracted from the tissue, and reapplied to other wounds. For example, fat tissue, bone marrow etc.
  • the invention allows the clinician to achieve one or more of the following benefits; a) minimise the donor skin area (improving patient rehab, speeding surgeon time, increasing chance of getting a quality graft), b) optimise the yield of active cells from the harvested tissue, increasing the surface area that can be covered, as well as the chances of a better healing outcome and reducing the scarring, c) reduce the processing time of the graft — reducing patient risk, increasing utilisation of therapy area, operating room, decreasing the time of staff in support, d) reduce the need for additional equipment — reducing cost, support staff, reducing introduction of secondary equipment into the area, reducing cleaning time, reducing the risk of infection, reducing expensive disposal such as batteries, and e) improve the patient outcome — with a better-quality healing, faster healing rate, less painful and speedier recovery, fewer complications.
  • a split thickness skin graft (STSG) is taken (epidermis and very thin section of dermis) using a range of devices such as manual and electric dermatomes or a Humby knife or silvers knife and this may vary between surgeons but the graft 2 should resemble a thickness similar to that shown in Figure 1.
  • Tissue cutting before enzyme treatment RenovaCare cuts the pieces smaller stating specifically only that “The skin tissue was cut into large connected, comb-like strips using forceps and a scalpel” and Avita leave the skin as a whole piece in tact with no prior cutting.
  • the tissue is cut into 1-2 mm 2 using a long blade 4 (5-7 cm), this makes it very quick to cut the tissue up and also we use an anti-blunting surface (like a plastic chopping board material) 6, that others to the best of our knowledge do not use.
  • RenovaCare places tissue pieces into two separate enzymes each at a temperature of 37°C, the first is dispase (bacterial) for 40-mins and the second is trypsin (porcine) for 15-minutes thus they need a 37°C incubator on-site and together a total time of around 75-minutes.
  • RenovaCare centrifuge the cells into a pellet, aspirate the solution, then re-suspend the pellet (hence this requires specialist instrumentation on site — including centrifuge, aspiration system).
  • Avita use porcine trypsin (porcine) for 15-45 minutes at 37°C, they use an electric system integrated into the kit (integrated heating element) that is operated by AA batteries to heat the enzyme solution to this temperature. They reduce enzyme activity by dilution of the enzymatic solution.
  • VeritaCellTM uses human (non-animal) recombinant lOx trypsin for 20-30 minutes at room operating theatre temperature (we have used 22°C routinely).
  • our method is faster, is relevant to operating theatre temperatures, no heating is required, has a human enzyme and this trypsin suspension can be inactivated better than porcine trypsin using dilution.
  • RenovaCare do not state if they mince after trypsinisation, however Avita physically scrape the tissue.
  • Straining cells to remove unwanted tissue RenovaCare isolates cells from digested unwanted tissue (mainly dermis) mixed together in solution by passing it through a 40 pm pore size strainer and then again by centrifuging them and re-suspending the pellet in buffer (as mentioned above). Avita pass digested unwanted tissue containing solution through a 100 pm pore size strainer only and this completes their isolation of cells.
  • VeritaCell uses a 5 pm filter first draining off excess enzyme and removing any dermal fibres that remain. The applicant has evidence that these fibres 8 stick with great affinity to cell isolate (see Figure 2). Because this is such a small pore size, Veritacell uses negative pressure to pull liquid and cells through the 5 pm filter 10(see Figure 3), leaving the dermal fibres (long strings of extra-cellular matrix proteins) behind which provides a better yield and a uniform ‘non-aggregated’ cell suspension 12 (see Figure 4). The Applicant has found that cell recovery and proliferation (growth) is far superior using this method and it is unique from RenovaCare’s or Avita’s method in that negative pressure and a very small pore size is used, based on fundamental cell biology knowledge of cell behaviour.
  • Solution used for cell suspension Cells are placed into a buffered solution by all competitors because this maintains the correct physiological pH and Osmolality.
  • RenovaCare use a technologically advanced electric spray gun to disperse the suspension on the wound and Avita uses a simple spray cap and tube (the same as a perfume bottle).
  • VeritaCell aims to simply drip gentiy a solution on the necessary areas of the wound using a simple syringe. Spraying is an unnecessary process that results in liquid and consequently cellular material loss.
  • _Vcntaccirs kit components are stable for one year in temperatures between 4-22°C and no special lab equipment is needed. Also the VeritaCell kit can produce better yields if the surgeon/nurse is prepared to soak the tissue pieces in enzyme in the fridge or on ice (if available) for between 60- minutes to 6 hours to help the enzyme penetrate the tissue, whilst the enzyme remains mostiy inactive. They then can transfer the skin to theatre temperature to accelerate the digestion of the tissue. This however is not compulsory, but is preferred if they have the time for better yields and higher numbers of viable cells. (for example, would be recommended for burns patients).
  • the kit is flexible in terms of speed depending on the desired outcome of the user/ surgeon and their application.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Dermatology (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Prostheses (AREA)

Abstract

L'invention concerne un procédé d'isolement de cellules à utiliser pour la réparation de plaies, ledit procédé comprenant une étape de prélèvement d'une greffe de peau sur un patient, ladite greffe étant ensuite exposée à de la trypsine recombinante humaine (non animale).
PCT/EP2020/075653 2019-09-13 2020-09-14 Procédé et appareil pour l'isolement de cellules et cellules isolées pour la cicatrisation Ceased WO2021048441A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP20788982.5A EP4028035A1 (fr) 2019-09-13 2020-09-14 Procédé et appareil pour l'isolement de cellules et cellules isolées pour la cicatrisation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1913264.6 2019-09-13
GB201913264A GB201913264D0 (en) 2019-09-13 2019-09-13 Method and apparatus for cell isolation

Publications (1)

Publication Number Publication Date
WO2021048441A1 true WO2021048441A1 (fr) 2021-03-18

Family

ID=68315392

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2020/075653 Ceased WO2021048441A1 (fr) 2019-09-13 2020-09-14 Procédé et appareil pour l'isolement de cellules et cellules isolées pour la cicatrisation

Country Status (3)

Country Link
EP (1) EP4028035A1 (fr)
GB (1) GB201913264D0 (fr)
WO (1) WO2021048441A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11987787B2 (en) 2020-07-22 2024-05-21 AVITA Medical Americas, LLC Devices, methods, and kits for preparing a cell suspension
US12180456B2 (en) 2022-12-27 2024-12-31 AVITA Medical Americas, LLC Tissue healing

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7351549B2 (en) * 2000-01-24 2008-04-01 Polymun Scientific Immunbiologische Forschung Gmbh Method for the manufacture of recombinant trypsin
EP2343079A2 (fr) * 2001-02-07 2011-07-13 McComb Foundation Inc. Appareil pour la préparation de suspensions cellulaires
WO2013142254A1 (fr) * 2012-03-22 2013-09-26 Avita Medical Ltd. Suspension cellulaire et son utilisation
WO2019066663A1 (fr) * 2017-09-30 2019-04-04 Auckland Uniservices Limited Procédé d'obtention de cellules à partir d'un dispositif de digestion de peau entière

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7351549B2 (en) * 2000-01-24 2008-04-01 Polymun Scientific Immunbiologische Forschung Gmbh Method for the manufacture of recombinant trypsin
EP2343079A2 (fr) * 2001-02-07 2011-07-13 McComb Foundation Inc. Appareil pour la préparation de suspensions cellulaires
WO2013142254A1 (fr) * 2012-03-22 2013-09-26 Avita Medical Ltd. Suspension cellulaire et son utilisation
WO2019066663A1 (fr) * 2017-09-30 2019-04-04 Auckland Uniservices Limited Procédé d'obtention de cellules à partir d'un dispositif de digestion de peau entière

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GAIL DUTTON: "Spray-On Stem Cells Can Step Up Healing : RenovaCare's CellMist System Promises to Accelerate the Healing of Burns and Wounds", GENETIC ENGINEERING & BIOTECHNOLOGY NEWS, vol. 37, no. 8, 15 April 2017 (2017-04-15), US, pages 6 - 7, XP055761513, ISSN: 1935-472X, DOI: 10.1089/gen.37.08.04 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11987787B2 (en) 2020-07-22 2024-05-21 AVITA Medical Americas, LLC Devices, methods, and kits for preparing a cell suspension
US12180456B2 (en) 2022-12-27 2024-12-31 AVITA Medical Americas, LLC Tissue healing
US12270019B2 (en) 2022-12-27 2025-04-08 AVITA Medical Americas, LLC Automated method
US12281298B2 (en) 2022-12-27 2025-04-22 AVITA Medical Americas, LLC System for automated preparation of a regenerative epidermal suspension and related methods of use

Also Published As

Publication number Publication date
EP4028035A1 (fr) 2022-07-20
GB201913264D0 (en) 2019-10-30

Similar Documents

Publication Publication Date Title
US10631974B2 (en) Cell suspension preparation technique and device
Mutalik et al. Surgical management of stable vitiligo: a review with personal experience
JP6042377B2 (ja) 細胞懸濁液の作製方法及び細胞懸濁液
EP1226234B1 (fr) Transplantation de cheveux
WO2021048441A1 (fr) Procédé et appareil pour l'isolement de cellules et cellules isolées pour la cicatrisation
CN104232475B (zh) 一种快速即时分离人皮肤表皮细胞、黑色素细胞和成纤维细胞的装置及方法
AU2002227802A1 (en) Cell suspension preparation technique and device
CN105154388B (zh) 一种皮肤角质细胞分离、培养方法
CN112522180B (zh) 一种人头皮毛囊单细胞悬液及其制备方法和用途
CN112961820A (zh) 一种皮肤组织采集、提取并制备细胞悬液的方法和装置
US20200255808A1 (en) Method for producing a skin equivalent, and use thereof for in vitro tests and in vivo transplants
Darmaputra Autologous Mini Punch Graft in Vitiligo: A Narrative Review
HK40018126A (en) Cell suspension preparation method
CN107921069B (zh) 脂肪耗尽的脂肪组织和其制备装置和方法
CN114457009A (zh) 一种干细胞提取物的制备方法及其应用
HK1212885B (en) Cell suspension preparation device
Kumar et al. Grafting of autologous non-cultured melanocytes for the treatment of Vitiligo: a pilot study
HK1057713B (en) Cell suspension preparation technique and use

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20788982

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020788982

Country of ref document: EP

Effective date: 20220413