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WO1999023199A1 - Systeme clos de traitement de tissus primaires - Google Patents

Systeme clos de traitement de tissus primaires Download PDF

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Publication number
WO1999023199A1
WO1999023199A1 PCT/US1998/022563 US9822563W WO9923199A1 WO 1999023199 A1 WO1999023199 A1 WO 1999023199A1 US 9822563 W US9822563 W US 9822563W WO 9923199 A1 WO9923199 A1 WO 9923199A1
Authority
WO
WIPO (PCT)
Prior art keywords
bag
tissue
dissociation
cells
kit
Prior art date
Application number
PCT/US1998/022563
Other languages
English (en)
Inventor
Mark B. Jones
David A. Maryanov
Robin L. Geller
Steven K. Neuenfeldt
Original Assignee
Baxter International Inc.
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 Baxter International Inc. filed Critical Baxter International Inc.
Publication of WO1999023199A1 publication Critical patent/WO1999023199A1/fr

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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
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting

Definitions

  • This invention relates to kits and methods for dissociating primary tumors and other tissues into viable single cell suspensions while maintaining an aseptic environment.
  • Cells which secrete therapeutically useful substances can be used in a number of applications, including gene therapy for treating cancer and other diseases.
  • the isolation of tumors and other tissues provides a source of cells for genetic engineering and, subsequently, for gene therapy purposes.
  • methods to effectively dissociate the isolated tissue mass into viable single cells or cell clusters are necessary in order to utilize these cell sources.
  • Processing of primary tumor biopsies to yield heterogeneous single cell suspensions currently takes several hours and usually requires enzymatic chemical digestion and shear mechanical dissociation (Freshney R.I. Disaggregation of the Tissue and Primary Culture. Chapter 11 in Culture of Animal Cells A Manual of Basic Technique, 99-118. New York: Alan R. Liss, Inc., 1983).
  • the present invention provides an improved method and a kit for preparing
  • An aseptic environment is maintained by
  • tissue samples dissociating tissue samples in a closed environment.
  • tissue samples dissociating tissue samples in a closed environment.
  • samples includes both tumorigenic and nontumorigenic tissues.
  • the term tumorigenic and nontumorigenic tissues includes both tumorigenic and nontumorigenic tissues.
  • closed environment refers to a container that is closed once the tissue sample is
  • the present invention provides a kit for tissue processing
  • tissue debris and a container or chamber to collect the processed cellular materials
  • the solid particles comprise beads selected from the group
  • bag is transparent or translucent.
  • flexible container comprises a
  • the dissociation bag is separated into two chambers by the
  • a collection bag is separated from the
  • dissociation bag by the filter, and the dissociation bag is used for processing the tissue
  • the kit further comprises a wash bag containing medium, inhibitors, or
  • the present invention provides a method for dissociating
  • the method comprises placing
  • tissue-compatible liquid in a flexible container which is in fluid connection with a
  • the solid particles comprise beads selected from the group
  • the flexible container comprises a
  • dissociation bag comprising an access port for introducing tissue and tissue compatible medium into the dissociation bag and a filter for separating, via gravity
  • the dissociation bag is transparent or
  • the dissociation bag is separated into two
  • chambers by the filter with one chamber used to process the tissue sample and the
  • a collection bag is separated from the dissociation bag
  • the dissociation bag is used for processing the tissue sample and the
  • the method further comprises a wash bag containing medium
  • inhibitors or enzymes, which is connected to the dissociation bag or to the collection
  • Figure 1 schematically shows the glass beads-tumor sample interface.
  • Figure 2 shows a prototype tumor processing kit.
  • Figure 3 shows a microscopic view of a histological section of processed primary
  • FIG. 4 shows a microscopic view of a histological section of cultured MCA-38
  • tissue samples includes both
  • the tissue processing kit comprises a flexible container for receiving
  • tissue in a tissue compatible medium solid particles for grinding the tissue in the
  • the flexible container comprises an access port for introducing the
  • tissue and the tissue compatible medium into the container and a filter for separating
  • the flexible container comprises a
  • Dissociation bag that is transparent or translucent. Dissociation bag material properties
  • Suitable dissociation bag materials include hydrocarbon
  • plastics and elastomers such as polyolefins (polyethylene, polypropylene), natural
  • rubber or synthetic elastomers such as polystyrene, acrylic, polyvinyl esters (polyvinyl alcohol, polyvinyl acetyl, ethylene vinyl acetate copolymer);
  • heterochain thermoplastics such as polyamides, polyester, polyether and cellulosic
  • thermosetting resins such as phenolic, amino, unsaturated polyester
  • the dissociation bag will contain epoxy, polyurethane and silicone.
  • the dissociation bag will contain epoxy, polyurethane and silicone.
  • plastic e.g. polyethylene
  • venting material like Tyvek® for aeration. This allows an operator to apply
  • the operator is able to visually identify larger tumor sections, and localized particle
  • a collection bag separated from the dissociation
  • bag by an in-line filter is used for collecting the single cells or clusters of cells.
  • the dissociation bag is separated into two chambers by the filter, with
  • one chamber used to process the tissue sample and the other chamber to collect the
  • filtration is accomplished via gravity force.
  • kit further comprises providing wash solutions
  • clusters from a wash bag containing medium, inhibitors, or enzymes, which is in fluid
  • wash bags comprise the same materials and properties as the dissociation bag.
  • the solid particles of the kit preferably comprise glass, ceramic, natural
  • the solid particles are preferably greater in diameter
  • the solid particles generally range in
  • the beads range in diameter from between about 100 mm to about 250 mm. In a most
  • the beads are about 165 mm in diameter.
  • the ratio is about 165 mm in diameter.
  • the size of the beads can be varied, thus
  • the tissue mass is dissociated into cells and tumor
  • bag 14 the contents of which can be centrifuged if a cell pellet is required.
  • filtration can be accomplished by other means, such as vacuum or pump
  • a wash bag 16 is optional and may contain wash solutions, enzymes or
  • solutions include, without limitation, cell specific growth media, phosphate buffered
  • PBS saline
  • Plasmalyte® Plasmalyte®
  • non-buffered saline saline
  • Suitable enzyme inhibitors depend
  • enzymes used for dissociation include, but are not limited to serum and specific enzyme inhibitors such as leupeptin and antipain.
  • serum and specific enzyme inhibitors such as leupeptin and antipain.
  • enzyme inhibitors are added to the dissociation bag 8 via tubing 18.
  • the dissociation bag 8 via tubing 18.
  • wash bag 16 could be similarly connected to the collection bag 14 to provide wash
  • the exact size and volume of the dissociation bag to be used will vary with the
  • the dissociation bag must be of a size
  • the bag must be proportionately smaller so that the sample is not lost in the
  • the separating filter 10 used will vary depending on the size of the
  • separating filter 10 must allow the dissociated cells or cell clusters to pass from the
  • the size of the collection bag will vary depending on
  • the size of the tissue mass to be dissociated For example, if the tissue mass is very
  • the present invention teaches a method for producing single
  • tissue compatible liquid tissue compatible liquid.
  • the solid particles in the container are then externally
  • the single cells or clusters of cells are subsequently separated and collected.
  • an effective grinding amount of solid particles means that number, type and
  • the method allows greater surface areas of tissue to be dissociated at any one time
  • closed environment refers to a container that is closed once the tumor is placed
  • the tissue compatible liquid may comprise any suitable liquid which maintains
  • Suitable media include,
  • cell specific growth media without limitation, cell specific growth media, phosphate-buffered saline ('TBS),
  • Plasmalyte® and non-buffered saline The method of the present invention utilizes mechanical dissociation by solid
  • solid particles 2 within a flexible container 4 mimic
  • the solid particles In a preferred embodiment of the present invention, the solid particles
  • the size of the beads can be varied, thus varying the resulting void
  • the beads are preferably greater in diameter than the single
  • the solid particles generally range in
  • the beads range in diameter from between about 100 mm to about 250 mm. In a most
  • the beads are about 165 mm in diameter.
  • the ratio of beads to cell mass should be between 0.1 and 10. As the number of beads used is decreased,
  • the flexible container preferably comprises a dissociation bag comprising a
  • reversibly sealable access port for introducing tissue, beads and tissue compatible liquid
  • portal and cap designed with a mechanical seal to isolate the bag contents.
  • the dissociation bag is separated into two chambers by the
  • a collection bag is separated from the
  • the dissociation bag is used for processing the tissue
  • the tissue sample, the suspension of dissociated tissue is drained by gravity through
  • the method further comprises providing wash
  • clusters from a wash bag containing medium, inhibitors, or enzymes, which is in fluid
  • the tumor processing procedure in this Example was used in the following
  • Tissue processing can be carried out at almost any temperature, limited only by the
  • the cell suspension was evaluated for viability (trypan blue exclusion) by
  • Example 2 Dissociation of MCA-38 primary tumor without filtration
  • mice syngeneic mouse carcinoma cells (MCA-38) into C57B6 mice (Harlan). The cells
  • Tumor was
  • the tumor processing system contained the following components (refer to).
  • the cut opening in the dissociation bag was heat sealed with a
  • a sterile tube connector (Terumo) was used to connect the tubing of
  • the tubing of the collection bag and the dissociation bag were connected. Approximately 50 ml of the cell suspension was
  • Example 3 Dissociation of MCA-38 primary tumor with filtration
  • the tumor processing system contained the following components (refer to Figure 2):
  • Resected tumor was enzymatically digested and mechanically dissociated as
  • Example 2 An in-line filter was connected to the collection bag with a sterile tube connector (Terumo). The in-line filter was connected by sterile spike
  • dissociation bag successfully dissociated the tumor into a single cell suspension
  • the filter allowed for easy separation of the cell suspension from the remaining tissue
  • Example 4 Dissociation of primary human glioblastoma
  • the in-line filter was connected to the collection bag with a sterile tube
  • B16 melanoma tumor derived from cells obtained from the American Type Tissue Collection (ATCC; Accession No. CRL6323) was initiated and harvested as
  • the tumor processing system contained the following
  • the cut opening in the dissociation bag was heat sealed with a hand held
  • the in-line filter was connected to the collection bag with a sterile tube
  • B16 melanoma tumor was initiated and harvested as described in Example 2.
  • Example 7 Dissociation of RIP-Tag insulinoma tumor
  • the tumor processing system the tumor processing system.
  • Example 8 Dissociation of MCA-38 primary tumor using a different dissociation
  • the tumor processing system contained the following components (refer to Figure
  • Example 9 Dissociation of RIP-Tag insulinoma primary tumor
  • B 16 melanoma tumor was initiated and harvested as described in Example 2.
  • the tumor processing system contained the following components (refer to Figure 2):
  • Example 5 Samples were dissociated with glass beads or stainless steel beads to
  • Example 11 Tumorigenic potential of processed B 16 primary tumor cells
  • Example 12 Survival and Proliferation of processed MCA-38 primary tumor cells This experiment was conducted to determine if processed MCA-38 primary
  • mice mouse carcinoma cells (MCA-38) into C57B6 mice (Harlan). After approximately 10 days
  • the tumor processing system contained the following components (refer to Figure 2):
  • MCA-38 cells were dissociated and collected with the same procedure as Example 5. Briefly, one corner of component A was cut open with scissors. Resected
  • the bag was agitated to dissociate the tumor.
  • Component B was connected to component C with a sterile tube connector
  • Component B in-line filter was connected to component A and
  • saline at a concentration of lxlO 7 cells/20 ⁇ l of saline.
  • the loaded devices were placed in a 150 x 15 mm petri dish
  • TheraCyteTM devices explanted at fourteen days is shown in Figures 3 and 4.
  • Processed MCA-38 primary tumor looked approximately the same as cultured MCA-

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

Abstract

L'invention porte sur des procédés et trousses de traitement de tumeurs en milieu clos par dissection par clivage à l'aide de particules solides. Le temps de dissociation des tissus est notablement moindre qu'avec les procédés usuels (minutes au lieu d'heures), et il est possible de dissocier de plus grandes surfaces de tumeurs par unité de temps en raison de l'accroissement de la surface de contact billes/tumeur. L'invention a permis de dissocier de manière reproductible des tumeurs primaires en suspensions viables de cellules hétérogènes isolées, tout en maintenant la fonction cellulaire.
PCT/US1998/022563 1997-10-31 1998-10-26 Systeme clos de traitement de tissus primaires WO1999023199A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US96208397A 1997-10-31 1997-10-31
US08/962,083 1997-10-31

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WO1999023199A1 true WO1999023199A1 (fr) 1999-05-14

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8785193B2 (en) 2006-09-14 2014-07-22 The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Dissection tool and methods of use
WO2018130845A1 (fr) * 2017-01-13 2018-07-19 Cellular Therapeutics Ltd Procédé, kit et dispositif de traitement aseptique de tissus
CN111304081A (zh) * 2013-03-14 2020-06-19 埃维塔医疗有限公司 用于组织处理以及由其制备细胞悬液的系统和方法
WO2021123832A1 (fr) * 2019-12-20 2021-06-24 Instil Bio (Uk) Limited Dispositifs et procédés d'isolement de lymphocytes infiltrant les tumeurs et leurs utilisations
US11530386B2 (en) 2015-12-15 2022-12-20 Instil Bio (Uk) Limited Cells expressing recombinant growth factor receptors
US11879121B2 (en) 2021-04-20 2024-01-23 CisNovo Tissue disaggregation system and methods
US12180456B2 (en) 2022-12-27 2024-12-31 AVITA Medical Americas, LLC Tissue healing

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2841339A (en) * 1954-03-25 1958-07-01 Dale T Gilmore Dehydrating and pulverizing machine
US3172546A (en) * 1961-05-19 1965-03-09 Union Carbide Corp Size reduction of biological substances
WO1988009667A1 (fr) * 1987-06-08 1988-12-15 Washington University Procede d'isolement d'agglomerats de sous-types de cellules a partir d'organes
EP0540390A1 (fr) * 1991-10-29 1993-05-05 Daniel Georges Couverchel Matériau comportant des pigments notamment luminescents, et son procédé d'obtention
WO1995002458A1 (fr) * 1993-07-14 1995-01-26 Shimakyu Chemical Co., Ltd. Sac d'ecrasement et de dispersion
WO1995002457A1 (fr) * 1992-01-16 1995-01-26 Shimakyu Chemical Co., Ltd. Sac a dechiqueter et disseminer
WO1996036694A1 (fr) * 1995-05-17 1996-11-21 Neocrin Company Isolement de cellules d'un tissu d'organe par sonication
US5679565A (en) * 1995-04-10 1997-10-21 The Regents Of The University Of California Method of preserving pancreatic islets

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2841339A (en) * 1954-03-25 1958-07-01 Dale T Gilmore Dehydrating and pulverizing machine
US3172546A (en) * 1961-05-19 1965-03-09 Union Carbide Corp Size reduction of biological substances
WO1988009667A1 (fr) * 1987-06-08 1988-12-15 Washington University Procede d'isolement d'agglomerats de sous-types de cellules a partir d'organes
EP0540390A1 (fr) * 1991-10-29 1993-05-05 Daniel Georges Couverchel Matériau comportant des pigments notamment luminescents, et son procédé d'obtention
WO1995002457A1 (fr) * 1992-01-16 1995-01-26 Shimakyu Chemical Co., Ltd. Sac a dechiqueter et disseminer
WO1995002458A1 (fr) * 1993-07-14 1995-01-26 Shimakyu Chemical Co., Ltd. Sac d'ecrasement et de dispersion
EP0709136A1 (fr) * 1993-07-14 1996-05-01 Shimakyu Chemical Co., Ltd. Sac d'ecrasement et de dispersion
US5679565A (en) * 1995-04-10 1997-10-21 The Regents Of The University Of California Method of preserving pancreatic islets
WO1996036694A1 (fr) * 1995-05-17 1996-11-21 Neocrin Company Isolement de cellules d'un tissu d'organe par sonication

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8785193B2 (en) 2006-09-14 2014-07-22 The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Dissection tool and methods of use
EP4455258A3 (fr) * 2013-03-14 2025-01-15 AVITA Medical Americas, LLC Systèmes et procédés de traitement de tissu et de préparation d'une suspension cellulaire à partir de ceux-ci
CN111304081A (zh) * 2013-03-14 2020-06-19 埃维塔医疗有限公司 用于组织处理以及由其制备细胞悬液的系统和方法
EP3674396A1 (fr) * 2013-03-14 2020-07-01 Avita Medical Ltd. Systèmes et procédés pour le traitement de tissus et préparation de suspension cellulaire à partir de ceux-ci
US11124752B2 (en) 2013-03-14 2021-09-21 Avita Medical Ltd Systems and methods for tissue processing and preparation of cell suspension therefrom
US11530386B2 (en) 2015-12-15 2022-12-20 Instil Bio (Uk) Limited Cells expressing recombinant growth factor receptors
US20220290094A1 (en) * 2017-01-13 2022-09-15 Instil Bio (Uk) Limited Aseptic tissue processing method, kit and device
US20220364042A1 (en) * 2017-01-13 2022-11-17 Instil Bio (Uk) Limited Aseptic tissue processing method, kit and device
US12173271B2 (en) * 2017-01-13 2024-12-24 Instil Bio (Uk) Limited Aseptic tissue processing method, kit and device
US11618877B2 (en) 2017-01-13 2023-04-04 Instil Bio (Uk) Limited Aseptic tissue processing method, kit and device
US11618878B2 (en) * 2017-01-13 2023-04-04 Instil Bio (Uk) Limited Aseptic tissue processing method, kit and device
US20230235269A1 (en) * 2017-01-13 2023-07-27 Instil Bio (Uk) Limited Aseptic tissue processing method, kit and device
WO2018130845A1 (fr) * 2017-01-13 2018-07-19 Cellular Therapeutics Ltd Procédé, kit et dispositif de traitement aseptique de tissus
WO2021123832A1 (fr) * 2019-12-20 2021-06-24 Instil Bio (Uk) Limited Dispositifs et procédés d'isolement de lymphocytes infiltrant les tumeurs et leurs utilisations
US11767510B2 (en) 2019-12-20 2023-09-26 Instil Bio (Uk) Limited Devices and methods for isolating tumor infiltrating lymphocytes and uses thereof
US11879121B2 (en) 2021-04-20 2024-01-23 CisNovo Tissue disaggregation system and methods
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

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