WO2012079749A1 - Process for making cell suspension - Google Patents

Process for making cell suspension 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
French (fr)
Inventor
Joseph T. Delaney
Albert R. Liberski
Hendrik SCHÄFER
Ulrich Ditmar Schubert
Original Assignee
Stichting Dutch Polymer Institute
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Publication of WO2012079749A1 publication Critical patent/WO2012079749A1/en

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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

The present invention relates to a process for making a suspension of single-cell beads each comprising a reversible gel matrix and one cell. The process comprises the steps of (A) preparing a curable liquid containing the cells; (B) dispensing the curable liquid in droplets into a curing environment to cure the curable liquid by ink-jet technology to form beads; and (C) sorting the single-cell beads from the beads obtained from step (B) by a flow cytometer.

Description

T EP2011/006304
- 1 -
PROCESS FOR MAKING CELL SUSPENSION
The present invention relates to a process for making a suspension of cells. In particular, 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
applications in both research and clinical practice. 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.
Flow cytometry are commonly used, but it is not without its
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.
H. G. Monbouquette et. Al, Biotechnology. (TV. Y.), 1988, 6, 1076-
1079 describes a process in which an 18 gauge needle was used to dispense sodium alginate/cell suspensions into calcium chloride, yielding large, multicellular beads in the range of 2.5 to 3 mm in diameter, which were analyzed using a microfluorimetry device. This publication does not disclose sorting of the beads.
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. The T EP2011/006304
- 2 -
beads in the selection gate (more than 10 pm) was analyzed by flow cytometry to confirm the screen efficiency of the system.
There is a need in the art for improvements for a process for making a suspension of cells using flow cytometry.
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.
Accordingly, 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:
(A) preparing a curable liquid containing the cells;
(B) dispensing the curable liquid in droplets into a curing environment to cure the curable liquid by ink-jet technology to form beads; and
(C) sorting the single-cell beads from the beads obtained from step (B) by a flow cytometer.
It is noted that 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.
It is further noted that 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.
The inventors have realized that 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. The inventors have further realized that 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.
According to the present invention, 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.
Furthermore, 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.
In step (B), 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).
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. 8: 713-720 (1991) and Drug Development and Industrial Pharmacy (2001), 27(8), 825-"829. Besides these methods continuous inkjet techniques can be used, for example binary deflection-, multiple deflection-, Hertz- and microdot-inkjet techniques.
In the context of the present invention the term "cell" denotes all types of cells which can associate with microcarrier beads as described above. Thus, 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. However, 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. Thus, one may e.g. use a fluorescent marker that specifically interacts with the cell type to be detected. In one of its more simple embodiments, 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.
According to some embodiments of the present invention, the curable liquid comprises a solution of a hydrogel in a fluid sol state, and the curing environment comprises a liquid medium which gelates the curable liquid.
According to some embodiments of the present invention, the curable liquid is a solution of alginate, gelatine or carrageenan. Preferably, the curable liquid is an alginate solution.
According to some embodiments of the present invention, the liquid medium which gelates the curable liquid is an aqueous solution of CaCI2, BaCI2 or SrCI2. An aqueous solution of CaCI2 is preferred in many cases. However, some cell types (for example: stem cells, osteoblasts, myocytes, nerve cells, and cancer cells) are especially sensitive to calcium under certain conditions. Calcium can trigger 006304
- 5 -
specific differentiation of the stem cells under some conditions, as described in Adams et al., Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature 439(7076):599-603. Calcium concentration can control function and rate of osteoblast cells, thus entire bone formation depends on it, as described in 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. 2004;101 (14):5140-5145. For the effects of calcium to myocytes, nerve cells and cancer cells, Li et. Al, Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects. Am. J. Physiol. 1998 274(4 Pt 2):H1335-47, Catterall WA, Few AP. Calcium channel regulation and presynaptic plasticity. Neuron 2008;59(6):882-901 and Rodland KD, The role of the calcium-sensing receptor in cancer, Cell Calcium 2004 3;35(3):291-295 are referred respectively. In these cases where the combination of calcium and cell types and conditions trigger detrimental effects, other divalent and trivalent cations such as Ba2+ and Sr2* are used for optimal performance.
According to some embodiments of the present invention, 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.
According to some embodiments of the present invention, 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 μητι, more preferably 30 μιη, 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. For example, the concentration of the curable liquid may be 105 to 106 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.
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.
Hereinafter, the present invention is further illustrated referring to the attached schematic drawings in which:
Fig. 1 illustrates a curable liquid containing cells;
Fig. 2 illustrates a suspension of beads containing cells and
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. In this figure, 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. In Fig. 3a, 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.

Claims

A process for making a suspension of single-cell beads each comprising a reversible gel matrix and one cell, comprising the steps of:
(A) preparing a curable liquid containing the cells;
(B) dispensing the curable liquid in droplets into a curing environment to cure the curable liquid by ink-jet technology to form beads; and
(C) sorting the single-cell beads from the beads obtained from step (B) by a flow cytometer.
The process according to claim 1 , wherein the curable liquid comprises a solution of a hydrogel in a fluid sol state, and the curing environment comprises a liquid medium which gelates the curable liquid.
The process according to claim 2, wherein the curable liquid is solution of alginate, gelatine or carrageenan.
The process according to claim 2 or 3, wherein the liquid medium is is an aqueous solution of CaCI2, BaCI2 or SrCI2.
The process according to any one of claims 1-4, wherein the average volume of the droplets dispensed in step (B) is between 10 to 200 pL, preferably 24 to 84 pL, more preferably 40-50 pL.
The process according to any one of claims 1-5, wherein the average diameter of the beads is 1-250 μητι and has 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 process according to any one of claims 1-6, further comprising (D) sorting the single-cell beads from the beads obtained from step (C) by the flow cytometer.
The suspension of single-cell beads obtainable by the process according to any one of claims 1-7.
The suspension according to claim 8, wherein at least 95%, preferably at least 98%, more preferably 99%, more preferably 99.9%, of the beads are single- cell beads.
PCT/EP2011/006304 2010-12-16 2011-12-14 Process for making cell suspension WO2012079749A1 (en)

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NL10015708.0 2010-12-16

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000008212A1 (en) * 1998-08-07 2000-02-17 Cellay, Llc Gel microdrops in genetic analysis
JP2007111023A (en) * 2005-10-24 2007-05-10 Ajinomoto Co Inc Method for obtaining microorganism by using agarase
WO2008117195A2 (en) 2007-03-26 2008-10-02 Koninklijke Philips Electronics N. V. Use of microcarrier beads for detection and/or isolation of cells by flow cytometry
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 (en) * 1998-08-07 2000-02-17 Cellay, Llc Gel microdrops in genetic analysis
US7556928B2 (en) 2001-05-26 2009-07-07 One Cell Systems, Inc. Method of screening a population of cells
JP2007111023A (en) * 2005-10-24 2007-05-10 Ajinomoto Co Inc Method for obtaining microorganism by using agarase
WO2008117195A2 (en) 2007-03-26 2008-10-02 Koninklijke Philips Electronics N. V. Use of microcarrier beads for detection and/or isolation of cells by flow cytometry

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
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