WO2012079749A1 - Procédé pour réaliser une suspension de cellules - Google Patents

Procédé pour réaliser une suspension de cellules Download PDF

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Publication number
WO2012079749A1
WO2012079749A1 PCT/EP2011/006304 EP2011006304W WO2012079749A1 WO 2012079749 A1 WO2012079749 A1 WO 2012079749A1 EP 2011006304 W EP2011006304 W EP 2011006304W WO 2012079749 A1 WO2012079749 A1 WO 2012079749A1
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WO
WIPO (PCT)
Prior art keywords
beads
cells
cell
curable liquid
suspension
Prior art date
Application number
PCT/EP2011/006304
Other languages
English (en)
Inventor
Joseph T. Delaney
Albert R. Liberski
Hendrik SCHÄFER
Ulrich Ditmar Schubert
Original Assignee
Stichting Dutch Polymer Institute
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 Stichting Dutch Polymer Institute filed Critical Stichting Dutch Polymer Institute
Publication of WO2012079749A1 publication Critical patent/WO2012079749A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • the present invention relates to a process for making a suspension of cells.
  • the present invention relates to a process for making a suspension of cells using flow cytometry.
  • Flow cytometry is a technique for counting and examining
  • microscopic particles such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of health disorders, especially blood cancers, but has many other
  • a common variation is to physically sort particles based on their properties, so as to purify populations of interest.
  • Cells sorted by flow cytometry may be subjected to a wide range of subsequent experiments.
  • drawbacks One of the drawbacks is that the cells may be damaged during sorting. Another drawback is that it usually cannot be used to analyse multi-cell aggregates because of their large size, leading to instrument clogging.
  • WO2008/117 95 deals with the latter drawback by a method of detecting and/or isolating at least one cell comprising the steps of: providing at least one microcarrier bead; contacting said at least one microcarrier bead with at least one cell such that said at least one cell associates with said at least one microcarrier bead; detecting and/or isolating said cell-associated microcarrier bead.
  • Y. Zhou et.al, Appl. Microbiol. Biotechnol., 2009, 84, 375-382 describes a high-speed affinity screening system for yeast cells using flow cytometry adapted to screening yeast cells that display hydrolyzing enzymes.
  • the yeast cells were captured in micro-sized calcium alginate beads by a 'reverse micelle method' to prevent diffusion of hydrolyzed fluoresceng substrates.
  • Flow cytometry was used to determined the size of the beads in order to select the beads with yeast cells.
  • beads in the selection gate (more than 10 pm) was analyzed by flow cytometry to confirm the screen efficiency of the system.
  • An objective of the present invention is therefore to provide an improved process for making a suspension of cells where the above-mentioned and/or other needs in the art are met.
  • the present invention provides a process for making a suspension of single-cell beads each comprising a reversible gel matrix and one cell, comprising the steps of:
  • step (C) sorting the single-cell beads from the beads obtained from step (B) by a flow cytometer.
  • US7556928 describes a method of analzying a secreted protein from a cell encapsulated in a microdrop.
  • US7556928 describes that the microdrops are prepared by dispersing cells in liquefied biotinylated agarose (or other matrix molecules ) into an excess of a hydrophobic fluid to form an emulsion. The emulsion is transiently cooled, causing gelling. Once formed, microdrops are physically distinct and robust and can be removed from the oil into an aqueous medium by low speed centrifugation. Alternatively, microdrops can be formed by passing a mixture of liquefied gel and entities through a pulsating nozzle, such as the printhead of an inkjet printer.
  • US7556928 does not disclose dispensing curable liquid containing cells into a curing environement to cure the curable liquid by ink-jet technology to form beads.
  • WO00/08212 discloses a method of nucleic acid analysis.
  • the method comprises forming a population of gel microdrops encapsulating a population of biological entities.
  • the populations of gel microdrops may be formed by forming a preparation of biological entities in a liquid gel and passing the preparation through a pulsating orifice, which may be a component of an inkjet printer.
  • WO00/08212 does not disclose dispensing curable liquid containing cells into a curing environement to cure the curable liquid by ink-jet technology to form beads.
  • a suspension of single-cell beads is highly useful for further tests on the cells for obtaining statistical data from each individual cells.
  • the present invention is based on the realization that the combination of ink-jet technology and flow cytometry allows a highly efficient preparation of a cell suspension suitable for high- throughput experiments.
  • beads made by inkjet technology is highly suitable for being subjected to a flow cytometry. Due to the uniformity of the droplet sizes and volumes afforded by inkjet printing, beads can be made having a size that reduces the risk of clogging during flow cytometry.
  • a flow cytometer can be used to determine the number of cells in each bead and sorting beads accordingly, and this can directly be used for the preparation of a suspension of single-cell beads.
  • a further advantage of the process according to the present invention is that the uniformity of the droplet sizes by inkjet technology results in an increased probability of obtaining single-cell beads among beads made, thereby allowing efficient use of cells.
  • a suspension of beads is obtained in which each of the cells are separately protected by a reversible gel matrix.
  • the cells are protected both during the suspension is made and during the cells are subjected to further tests after the suspension is made.
  • the cells are protected by the gel matrix from the shear stresses encountered during the flow cytometry upon preparation of the suspension.
  • the cells are also less prone to dehydration/mechanical stress when subjected to further tests compared to the cells without the gel matrix.
  • the gel matrix covering each cell prevents the cells from having direct interaction with each other, preventing cells from e.g. aggregation. This is highly advantageous for further tests on the cells in that data may be obtained from each individual cells.
  • a further advantage of the beads obtained according to the process of the present invention is that it is highly suitable for recultivation.
  • the beads may be subjected to recultivation, and a bead which is a particularly interesting can be readily isolated from the others at a given timepoint without interfering with the viability of the rest.
  • the droplets may be ejected using several different ink-jet techniques, including both drop-on-demand techniques (e.g. thermo and piezoelectric- driven) as well as continuous jetting techniques (e.g. electrospraying and other methods based on Rayleigh jet breakup).
  • drop-on-demand techniques e.g. thermo and piezoelectric- driven
  • continuous jetting techniques e.g. electrospraying and other methods based on Rayleigh jet breakup.
  • Said drop-on-demand technique implies ejecting a discrete amount of liquid onto a specific location.
  • An important feature of such drop-on-demand technique for the process of this invention is its ability to eject uniform droplets. Any jetting technique can be used that allows control of the droplet size.
  • Examples of drop-on- demand techniques are drop-on-demand inkjet techniques, such as thermal, piezoelectric, electrostatic or acoustic inkjet techniques, or other techniques for creating microdroplets, such as pneumatic microvalve technology, double emulsion- solvent evaporation method or other emulsion methods. These technologies are known to those skilled in the art and are published, for example, in Tissue Engineering (2008), 14(1), 41-48, Pharm. Res.
  • continuous inkjet techniques can be used, for example binary deflection-, multiple deflection-, Hertz- and microdot-inkjet techniques.
  • cell denotes all types of cells which can associate with microcarrier beads as described above.
  • the term may relate to any type of cell that can be isolated from a biological sample such as bodily fluids including blood, plasma, urine, saliva etc.
  • samples may also comprise environmental samples taking from soils, lakes, rivers, plants etc.
  • Typical cell types which can be analysed by the methods in accordance with the invention include e.g. stem cells, progenitor cells, leukocytes, fibroblasts, tumour cells, sperm cells, fibroblasts, neuronal cells, hepatic cells and fetal cells.
  • Detection of the number of cells in the beads may be facilitated in known manners.
  • one may e.g. use a fluorescent marker that specifically interacts with the cell type to be detected.
  • the methods detect the cell species by fluorescent labelling. This can be achieved by addition of a simple fluorescent and cell-permeable dye, such as Hoechst stain.
  • the curable liquid comprises a solution of a hydrogel in a fluid sol state
  • the curing environment comprises a liquid medium which gelates the curable liquid
  • the curable liquid is a solution of alginate, gelatine or carrageenan.
  • the curable liquid is an alginate solution.
  • the liquid medium which gelates the curable liquid is an aqueous solution of CaCI 2 , BaCI 2 or SrCI 2 .
  • An aqueous solution of CaCI 2 is preferred in many cases.
  • some cell types for example: stem cells, osteoblasts, myocytes, nerve cells, and cancer cells
  • Calcium can trigger 006304
  • the average volume of the microdroplets of the curable liquid to be dispensed is between 10 to 200 pL, preferably 24 to 84 pL, more preferably 40 to 50 pL, as determined by the apparatus for ink-jet printing.
  • the beads obtained by step (B) have an average diameter of 1-250 pm. More preferably, the bead has a diameter of at most 50 ⁇ , more preferably 30 ⁇ , more preferably 20 pm. A particularly preferred range of the diameter is 5-15 pm. Preferably, the beads have a standard deviation of at most 20% of the average diameter, preferably 10%, more preferably 5%, even more preferably 1%, most preferably 0.1%. The largest dimension of the bead is herein called the diameter of the bead. The diameter of the beads may be determined e.g. by optical microscopy, light scattering spectroscopy, scanning electronic microscopy etc.
  • the concentration of the curable liquid can be decided according to specific needs, as long as the suspension can be deposited in microdroplets by inkjet technology.
  • the concentration of the curable liquid may be 10 5 to 10 6 cells/mL.
  • the concentration is determined by putting volumes of the suspension in sectors of a hemocytomer and counting the number of cells in each sector. A flow cytometer or particulate analyzer may also be used.
  • step (C) Since the cells are protected by the gel matrix, step (C) which may otherwise damage the cells may be repeated in order to improve the precision of the sorting. This feature leads to the possibility of 100% purity of cell culture after removing the gel matrix. Small and valuable cell populations can be sorted to a maximum of purity and a maximum of number of cells. This may be especially advantageous for blood cells, bone marrow cells, placenta cells, etc. Likewise, undesired cell subpopulations (e.g. cancer cells) can be rigorously removed. Accordingly, in some embodiments of the present invention, step (C) is repeated. It will be appreciated that the number of times step (C) is repeated depends on the use and the type of the cells, and may be once, twice, or even more.
  • Fig. 1 illustrates a curable liquid containing cells
  • Fig. 2 illustrates a suspension of beads containing cells
  • Fig. 3a-c illustrate a suspension of beads containing cells after sorting by a flow cytometry.
  • Fig. 1 illustrates a container containing a suspension of cells 20 in a curable liquid 10, provided by step A of the process of the present invention.
  • This suspension is to be dispensed in droplets into a curing environment by ink-jet technology (step B of the process of the present invention).
  • Fig. 2 illustrates a suspension of beads 11 each containing the cell 20.
  • the beads 11 is made by curing the curable liquid 10.
  • each of the beads comprises one to three cells or no cell.
  • This suspension is to be subjected to a flow cytometer (step C of the process of the present invention).
  • Fig. 3a-c each illustrates a suspension of beads 11 , resulting from sorting by the flow cytometry.
  • the suspension comprises beads 11 with no cells.
  • Fig. 3b shows a suspension of beads 11 each comprising one cell 20, which is the desired suspension according to the present invention.
  • Fig. 3c shows a suspension containing beads 1 each comprising two or more cells 20.

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  • Chemical & Material Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

La présente invention porte sur un procédé de réalisation d'une suspension de perles à cellule unique, comprenant chacune une matrice de gel réversible et une cellule. Le procédé comprend les étapes consistant (A) à préparer un liquide durcissable contenant les cellules ; (B) à distribuer le liquide durcissable en gouttelettes dans un environnement de durcissement pour durcir le liquide durcissable par une technologie à jet d'encre afin de former des perles ; et (C) à trier les perles à cellule unique parmi les perles obtenues à partir de l'étape (B) à l'aide d'un cytomètre de flux.
PCT/EP2011/006304 2010-12-16 2011-12-14 Procédé pour réaliser une suspension de cellules WO2012079749A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP10015708 2010-12-16
NL10015708.0 2010-12-16

Publications (1)

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WO2012079749A1 true WO2012079749A1 (fr) 2012-06-21

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000008212A1 (fr) * 1998-08-07 2000-02-17 Cellay, Llc Microgouttelettes de gel utilisees en analyse genetique
JP2007111023A (ja) * 2005-10-24 2007-05-10 Ajinomoto Co Inc アガラーゼを用いた微生物の取得方法
WO2008117195A2 (fr) 2007-03-26 2008-10-02 Koninklijke Philips Electronics N. V. Utilisation de billes micro-porteuses pour la détection et/ou l'isolement de cellules par cytométrie en flux et/ou diélectrophorèse
US7556928B2 (en) 2001-05-26 2009-07-07 One Cell Systems, Inc. Method of screening a population of cells

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000008212A1 (fr) * 1998-08-07 2000-02-17 Cellay, Llc Microgouttelettes de gel utilisees en analyse genetique
US7556928B2 (en) 2001-05-26 2009-07-07 One Cell Systems, Inc. Method of screening a population of cells
JP2007111023A (ja) * 2005-10-24 2007-05-10 Ajinomoto Co Inc アガラーゼを用いた微生物の取得方法
WO2008117195A2 (fr) 2007-03-26 2008-10-02 Koninklijke Philips Electronics N. V. Utilisation de billes micro-porteuses pour la détection et/ou l'isolement de cellules par cytométrie en flux et/ou diélectrophorèse

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
ADAMS ET AL.: "Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor", NATURE, vol. 439, no. 7076, pages 599 - 603
BOGEN K T ET AL: "Gel microdrop flow cytometry assay for low-dose studies of chemical and radiation cytotoxicity.", TOXICOLOGY 7 MAR 2001 LNKD- PUBMED:11246118, vol. 160, no. 1-3, 7 March 2001 (2001-03-07), pages 5 - 10, XP002638347, ISSN: 0300-483X *
CARROLL SILVIA ET AL: "The selection of high-producing cell lines using flow cytometry and cell sorting", EXPERT OPINION ON BIOLOGICAL THERAPY, ASHLEY, LONDON, GB, vol. 4, no. 11, 1 November 2004 (2004-11-01), pages 1821 - 1829, XP009075125, ISSN: 1471-2598, DOI: DOI:10.1517/14712598.4.11.1821 *
CATTERALL WA; FEW AP.: "Calcium channel regulation and presynaptic plasticity", NEURON, vol. 59, no. 6, 2008, pages 882 - 901
DRUG DEVELOPMENT AND INDUSTRIAL PHARMACY, vol. 27, no. 8, 2001, pages 825 - 829
DVORAK ET AL.: "Physiological changes in extracellular calcium concentration directly control osteoblast function in the absence of calciotropic hormones", PROC. NATL. ACAD. SCI. U.S.A., vol. 101, no. 14, 2004, pages 5140 - 5145, XP055087864, DOI: doi:10.1073/pnas.0306141101
H. G. MONBOUQUETTE, BIOTECHNOLOGY. (N. Y., vol. 6, 1988, pages 1076 - 1079
LI: "Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKlI effects", AM.J.PHYSIOL., vol. 274, 1998, pages H1335 - 47
NAKAMURA MAKOTO ET AL: "Biocompatible inkjet printing technique for designed seeding of individual living cells.", TISSUE ENGINEERING 2005 NOV-DEC LNKD- PUBMED:16411811, vol. 11, no. 11-12, November 2005 (2005-11-01), pages 1658 - 1666, XP002638345, ISSN: 1076-3279 *
PHARM. RES., vol. 8, 1991, pages 713 - 720
RINGEISEN BRADLEY R ET AL: "Jet-based methods to print living cells.", BIOTECHNOLOGY JOURNAL SEP 2006 LNKD- PUBMED:16895314, vol. 1, no. 9, September 2006 (2006-09-01), pages 930 - 948, XP002638346, ISSN: 1860-7314 *
RODLAND KD: "The role of the calcium-sensing receptor in cancer", CELL CALCIUM, vol. 35, no. 3, March 2004 (2004-03-01), pages 291 - 295
TAO XU ET AL: "High-Throughput Production of Single-Cell Microparticles Using an Inkjet Printing Technology", JOURNAL OF MANUFACTURING SCIENCE AND ENGINEERING, ASME INTERNATIONAL, NEW YORK, NY, US, vol. 130, 1 April 2008 (2008-04-01), pages 21017 - 1, XP008130891, ISSN: 1087-1357 *
TISSUE ENGINEERING, vol. 14, no. 1, 2008, pages 41 - 48
Y. ZHOU, APPL. MICROBIOL. BIOTECHNOL., vol. 84, 2009, pages 375 - 382

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