WO2024010763A1 - Préparation de cellules mononucléaires exemptes de plaquettes - Google Patents

Préparation de cellules mononucléaires exemptes de plaquettes Download PDF

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Publication number
WO2024010763A1
WO2024010763A1 PCT/US2023/026834 US2023026834W WO2024010763A1 WO 2024010763 A1 WO2024010763 A1 WO 2024010763A1 US 2023026834 W US2023026834 W US 2023026834W WO 2024010763 A1 WO2024010763 A1 WO 2024010763A1
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WIPO (PCT)
Prior art keywords
platelet
particles
platelets
pbmc
particle
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PCT/US2023/026834
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English (en)
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Thomas Russell
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Russell Biotech, Inc.
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Publication of WO2024010763A1 publication Critical patent/WO2024010763A1/fr

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    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • 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
    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles

Definitions

  • the invention provides novel means and methods for eliminating platelet contamination from peripheral blood mononuclear cells (PBMC)
  • PBMC peripheral blood mononuclear cells
  • the invention relates more specifically to kits and reagents to remove platelets from whole blood or PBMC using magnetic separation and/or gravity settling.
  • PBMC are comprised of desired lymphocytes and monocytes and are used routinely in biological research including immunological research.
  • PBMC are isolated from whole blood using Ficoll gradient centrifugation which removes granulocytes and red blood cells but not platelets yielding the desired lymphocytes and monocytes contaminated with platelets.
  • Numerous references (see ‘other publications’: [1-10]) provide means (primarily further centrifugation and washing steps) to remove the undesired platelets. Platelets are very sticky and as seen in the cited references often interfere with experiments requiring lymphocytes and or monocytes. Just a few examples are needed to reveal how undesirable platelets are.
  • the present invention provides methods and kits to remove platelets from whole blood, diluted whole blood or PBMC that remain part of the PBMC fraction following Ficoll density gradient centrifugation of whole blood.
  • the kits comprise reagents including nano/micro particles bound to antibodies that bind specifically to platelets. Once the anti-platelet particles are bound to the platelets means such as magnetic or gravity are used to remove the platelets from whole blood or PBMC yielding platelet free PBMC in high yield.
  • a preferred embodiment uses dense metallic nickel magnetic particles as described in US Patent 9435799 and US Patent 9739768 incorporated herein by reference.
  • the unique features of the particles (rapid magnetic separation times, rapid mixing times and the ability to work directly in undiluted whole blood with minimal loss of non-targeted cells (here PBMC) solve the problem and consequently yield platelet free PBMC.
  • Another preferred embodiment uses dense particles as described in US patent 5576185 incorporated herein by reference. Though larger than particles in ‘799 thus exhibiting slower reaction times the particles can be used to bind to platelets and then by simple gravity settling platelets are removed from whole blood or PBMC.
  • Another embodiment involves magnetic particles not made of metal but rather composed of metal oxides 50nm or less in diameter i.e. inorganic compounds (primarily iron oxides) imbedded in non-magnetic material as described in 5,411,863 (Miltenyi); 5,466,574 (Liberti); 4,654,267 (Ugelstad) and 4,707,523 (Chang). Magnetic particles available commercially are by and large made of these inorganic iron oxide magnetic particles and are referred to as superparamagnetic particles (Reference 12).
  • inorganic compounds primarily iron oxides
  • Figure 1 Properties of metal nickel particles used in a preferred embodiment of the invention.
  • Figure 2 Depicts the rapid kinetics seen with nickel particles used in a preferred embodiment of the invention.
  • the sample is undiluted whole blood.
  • the analysis is done on a Beckman Coulter 3 -part differential analyzer.
  • the granulocytes are removed using a particle of the invention coupled to CD15.
  • Figure 3 Depicts the high recovery of non-targeted cells seen with particles used in a preferred embodiment of the invention as compared to iron oxide superparamagnetic particles of the art.
  • Source BD Biosciences Catalog.
  • RBI Russell Biotech, Inc.
  • Figure 5 Properties of inorganic iron oxide particles defined as superparamagnetic particles that are currently available to the market.
  • the means to obtain platelet free PBMC as detailed in this invention comprise three separate methods all based on nano/microparticle technology.
  • the methods include two separate magnetic particles whereby the platelets are removed by a magnetic field and a dense particle whereby the platelets are removed by simple gravity sedimentation or accelerated gravity sedimentation (centrifugation).
  • the particles have bound thereto antiplatelet antibodies such as CD41 and CD61, but not limited to, that bind to platelets.
  • the antibodies used in this invention can be either polyclonal or monoclonal.
  • Such monoclonal antibodies are commercially available from a number of suppliers.
  • Mab are coupled to the particles of the invention by means known in the art including adsorption and numerous covalent coupling procedures.
  • the Mab can also be coupled to the particle using the biotin/streptavidin/avidin system as known in the art where usually the streptavidin/avidin is bound to the particle and the anti-platelet antibody is coupled to biotin.
  • Other coupling pairs known in the art are included in the invention.
  • the sample is whole blood (diluted or un-diluted) or PMBC.
  • Whole blood can be human or animal such as mouse or rat but not limited to any mammal having a platelet population in blood.
  • the preferred embodiment uses a dense magnetic metallic particle as described in Russell ‘799.
  • the particle can range in diameter from 0.3 micron to 3-4 micron with a preferred diameter around 1 micron.
  • the particle is dense having a density of 5-10 g/cc with a preferred density around 9 g/cc.
  • Any metal or metal alloy that is magnetic will function in the invention with a preferred metal being nickel.
  • Any metal commercially available that has the properties identified here such as from Sigma Aldrich but not limited to will function in the assay but the preferred nickel particle is that described in Russell ‘799.
  • Figure 1 details features of a preferred nickel particle that make it especially suited to remove platelets including the ability to work easily in undiluted whole blood rapidly with rapid magnetic separation times (Fig 2).
  • Figure 2 demonstrates the rapid removal of a cell population (granulocytes) directly from an un-diluted whole blood sample in very rapid mixing times using a magnetic particle as in this preferred embodiment. Another key feature of these particles is the ability to remove a desired cell population with high, almost quantitative recovery, of non-targeted cells (Fig 3).
  • undiluted or diluted whole blood will be mixed directly with the antiplatelet magnetic nickel particles by end-over-end mixing using a device from airbiotech, Inc. for volumes greater than 1 ml and vortexing for volumes equal to or less than 1 ml for the shortest time possible for binding to the platelets in the range of a few seconds to 10 minutes but not limited to.
  • the preferred rotation speed is around 15-30 rpm for 1 micron nickel particles.
  • the sample will be placed in a magnetic field depending on the sample volume for a few seconds up to 5 minutes. Suitable magnets are known in the art such as those from Dexter Magnetics.
  • the whole blood sample depleted of platelets will be removed while the sample chamber remains in the magnetic field and then separated on Ficoll to yield the much-desired platelet free PBMC in very high yield for further research studies.
  • any procedure known in the art for coupling antibody to the particles is incorporated herein i.e. adsorption or covalent coupling.
  • adsorption of the anti-platelet antibody directly to the nickel particle surface is simple and results in a very stable particle antibody complex when the isotype of the antibody is an IgM.
  • Anti-CD41 and anti-CD61 IgM isotypes are commercially available i.e. BioLegend. Any antibody isotype i.e. IgM or IgG but not limited thereto that adsorbs to the particle and effectively removes platelets is considered by the disclosure herein. Though experimental studies may be needed to obtain the optimal amount of antibody per particle routinely labelling at 2mg antibody /meter squared particle surface (surface area) is usually sufficient.
  • any magnetic particles with or without platelets bound thereto that may remain in the sample following placement in the magnetic field (carry over) will be removed following the Ficoll density gradient centrifugation because of the density of the nickel particle relative to cells thus yielding truly platelet free PBMC.
  • platelets can be removed, using particles of the invention, directly from PBMC. Any residual particles can then be removed by simple centrifugation at speeds that sediment the very dense particles but do not sediment PBMC. The speeds and times of centrifugation can be easily determined by means known in the art.
  • a second embodiment uses dense particles such as those described in U S Patent 5,576,185 incorporated herein by reference and sold by Novamet.
  • the particles are dense with densities in the range 5-10 g/cc with a preferred density around 9g/cc.
  • the particles can be any metal, metal alloy or ceramic that can settle by gravity in whole blood or PBMC.
  • the preferred particle diameter is 3-10 micron. Though any diameter particle that operates as described herein is covered by the invention.
  • the method involves dense particles with anti-platelet antibodies bound thereto as described herein (see preferred embodiment).
  • the particles can be added directly to whole blood or whole blood can be added to particles that are at the bottom of the mixing container.
  • the particles are mixed by end-over-end mixing at about 5-10 rpm for volumes greater than 1ml and by vortexing for volumes of 1 ml or less.
  • the sample container is simply placed in an upright position and the dense particles settle by gravity to the bottom of the tube.
  • the gravity settling can be accelerated by centrifugation at speeds that settle the dense particles but do not significantly settle the whole blood.
  • a magnet can be placed at the bottom of the tube to hold the particles in place while the platelet free blood is removed and then centrifuged over Ficoll to obtain platelet free PBMC.
  • any residual particles carried over, especially non-magnetic dense particles, will be removed by the centrifugation over Ficoll. Again, it is best to start with whole blood so that any residual dense particles can be removed in the Ficoll centrifugation step.
  • the starting material can be PBMC with removal of any residual dense particles due to carryover by a very rapid centrifugation step that leads to settled dense particles without settling of PBMC.
  • the nickel particles Prior to adding anti-platelet antibodies to nickel particles the nickel particles are weighed on a scale preferably in a hood while wearing a face mask. Though nickel has GRASS status, while working with nickel powder, one should avoid breathing dust from the particles. Weigh out the amount of nickel desired based on coating antibody at 2mg/meter squared surface area of the particle. The nickel particles are then heated at 250 degrees centigrade for 3 hours up to 3 days. This accomplishes the following important features: the particles are sterile if needed and an oxide coating covers the outer layer of the particle which significantly reduces the release of nickel ions from the particle and the nickel oxide surface can be used to covalently bind antibodies to the particles by means known in the art. Once the particles are in a buffer solution no more dust is present. A hood and/or mask are no longer required.
  • Another embodiment involves use of magnetic particles currently on the market supplied by numerous vendors including Miltenyi Biotec, BD Biosciences, TheromoFisher, EMD Millipore, PolySciences and Stem Cell Technologies to remove platelets from whole blood or PBMC. These particles differ from the magnetic metal particles of ‘799. They are superparamagnetic particles that are made up of inorganic metal oxides rather than metal. The oxide is usually iron oxide but any metal oxide that is magnetic falls under this embodiment. The metal oxide is para/ superparamagnetic because the metal oxide crystal is under 50 nm in diameter (see 5,466,574; 5,411,863; 4,654,267).
  • the metal oxide crystals are embedded in polymeric material by means known in the art to build particles of various diameters from 100 nanometers to 4.5 micron (see Figure 5).
  • the density of these particles is less than 2g/cc and thus mixing in undiluted whole blood is problematic and it is usually recommended that prior to adding magnetic particles to whole blood the blood is diluted and or red cells are lysed (Miltenyi Biotec).
  • Kits will be provided to the market for using the technology described in this invention to solve a major problem faced by researchers that of obtaining platelet free PBMC preparations.
  • the kits will include both consumable reagents and equipment.
  • the consumables will be particles (magnetic or dense magnetic or non-magnetic) of the invention in a suitable buffer such as Phosphate Buffered Saline (PBS) with protein such as 0.1% BSA, but not limited thereto. It is anticipated the particles will be bottled as 2ml and 5ml solutions but not limited thereto.
  • PBS Phosphate Buffered Saline
  • the kit will also contain a suitable rinsing buffer known in the art such as PBS with 0.1% BSA.
  • the equipment will be for a mixing means routinely purchased through Airbiotech or similar vendors and for magnetic particles a magnetic separation means though vendors such as Dextermag ⁇
  • a further embodiment of the present invention improves upon the described methods and includes a frozen fluid sample of whole blood or any biological sample containing PBMCs and not whole blood or PBMCs that had not been frozen.
  • the reason for the low viability following thawing of PBMCs may well be do the presence of varying number of platelets in the sample prior to freezing and or thawing. Since platelets are similar in density to PBMCs (14) they are enriched following density gradient centrifugation on FICOLL or other gradient material (14) used to isolate PBMCs. Therefore, a need exists for platelets to be removed before freezing and thawing PBMCs. As disclosed herein platelets are sticky and it makes sense to remove them. Reference 14 details a reason to remove them. The reference details the effect of platelets on mitochondrial(mt) DNA content of PBMCs. The copy number of mtDNA ranges from several tens of thousands to several hundreds of thousands depending on the tissue.
  • the copy number largely determines the cellular ATP production. Therefore, a decrease in mtDNA causes the cellular dysfunction leading to severe symptoms such as mtDNA depletion syndrome (14). Platelet contamination can lead to overestimation of mtDNA content suggesting that platelets be removed before mtDNA determination. Routinely platelets are removed by multiple washings that leads to PBMC loses.
  • the present invention describes procedures to remove platelets after thawing of PBMC samples.
  • Methods described herein leads to the effective removal of platelets from thawed PBMCs that were previously frozen before removal of platelets.
  • the thawed PBMCs are processed by the disclosure described herein for fresh PBMCs to yield platelet free thawed PBMCs.
  • platelets can be removed by gravity settling and magnetic separation. It is to be noted that particles of the art based on superparamagnetic particles though in operation are inferior to the particles of the invention.
  • platelet thawing may lead to breaking up of platelet particles into platelet fragments.
  • These possible platelet fragments still need to be removed. Their removal will not quantitate the presence of platelet fragments but ensures an enrichment method devoid of a certain amount of platelet fragments.
  • To deplete platelets that have not been frozen the above disclosure using anti-CD41 nickel particles is sufficient.
  • the present invention provides improved methodology.
  • PBMCs containing platelets and possible platelet fragments are treated with a three-bead combination to ensure a better chance of removing platelet fragments.
  • nickel magnetic or nickel gravity settling beads are made with not only CD41 but also with CD61 and CD36. Using this bead combination the odds of binding platelet fragments increases as a fragment may lack CD41 but then may have CD61 and/or CD36 present.
  • Patents/Patent Applications

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Abstract

Les plaquettes ont une densité similaire à celle des PBMC et sont donc des contaminants des PBMC isolées par centrifugation à gradient de densité de Ficoll, la procédure établie pour la préparation des PBMC. Les plaquettes sont très collantes et interfèrent souvent avec les expériences nécessitant des lymphocytes et/ou des monocytes (PBMC). Des procédés, des compositions et des kits sont présentés pour l'élimination rapide des plaquettes indésirables des PBMC, ce qui permet d'obtenir des PBMC exempts de plaquettes avec un rendement élevé. Le processus de sélection, dans le mode de réalisation préféré de l'invention, est fondé sur des particules métalliques magnétiques particulièrement adaptées à l'élimination des plaquettes du sang total ou des PBMC. La particule préférée est composée de nickel métal dans une gamme de taille préférée de 0,8 à 3,5 microns. Des agents anti-plaquettaires spécifiques sont liés aux particules de nickel. Les particules se mélangent rapidement à l'échantillon sans se lier de manière non spécifique à des cellules non ciblées. L'échantillon est ensuite placé dans un champ magnétique et les plaquettes liées aux particules magnétiques sont très rapidement éliminées, ce qui permet d'obtenir du sang total exempt de plaquettes ou des PBMC à très haut rendement en vue d'expériences biologiques ultérieures. Le procédé permet également l'élimination de fragments de plaquettes et de plaquettes de préparations de PBMC où des plaquettes peuvent être présentes avant la congélation ou après décongélation de la préparation de PBMC à l'aide d'une particule magnétique anti-plaquettaire combinée à trois billes.
PCT/US2023/026834 2022-07-07 2023-07-03 Préparation de cellules mononucléaires exemptes de plaquettes WO2024010763A1 (fr)

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US63/359,001 2022-07-07

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9739768B2 (en) * 2002-07-31 2017-08-22 Russell Biotech, Inc. Methods and reagents for improved selection of biological materials
US20180306817A1 (en) * 2015-10-07 2018-10-25 Sangui Bio Pty Ltd. Blood Preparation and Profiling
WO2018231373A1 (fr) * 2017-06-14 2018-12-20 Russell Biotech, Inc. Préparation de cellules mononucléaires exemptes de plaquettes
WO2020014175A1 (fr) * 2018-07-10 2020-01-16 Children's Medical Center Corporation Méthodes et compositions pour analyser des lignées de cellules progénitrices de mégacaryocytes immortalisées et particules de type plaquettes dérivées de celles-ci

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9739768B2 (en) * 2002-07-31 2017-08-22 Russell Biotech, Inc. Methods and reagents for improved selection of biological materials
US20180306817A1 (en) * 2015-10-07 2018-10-25 Sangui Bio Pty Ltd. Blood Preparation and Profiling
WO2018231373A1 (fr) * 2017-06-14 2018-12-20 Russell Biotech, Inc. Préparation de cellules mononucléaires exemptes de plaquettes
WO2020014175A1 (fr) * 2018-07-10 2020-01-16 Children's Medical Center Corporation Méthodes et compositions pour analyser des lignées de cellules progénitrices de mégacaryocytes immortalisées et particules de type plaquettes dérivées de celles-ci

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GAUTAM AARTI, DONOHUE DUNCAN, HOKE ALLISON, MILLER STACY ANN, SRINIVASAN SESHAMALINI, SOWE BINTU, DETWILER LEANNE, LYNCH JESSE, LE: "Investigating gene expression profiles of whole blood and peripheral blood mononuclear cells using multiple collection and processing methods", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 14, no. 12, 6 December 2019 (2019-12-06), US , pages e0225137, XP093128335, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0225137 *

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