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WO1994026104A1 - Procede d'isolement de cellules cibles - Google Patents

Procede d'isolement de cellules cibles Download PDF

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Publication number
WO1994026104A1
WO1994026104A1 PCT/US1994/005408 US9405408W WO9426104A1 WO 1994026104 A1 WO1994026104 A1 WO 1994026104A1 US 9405408 W US9405408 W US 9405408W WO 9426104 A1 WO9426104 A1 WO 9426104A1
Authority
WO
WIPO (PCT)
Prior art keywords
particles
cells
cell suspension
suspension
target cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1994/005408
Other languages
English (en)
Inventor
Edward F. Leonard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aprogenex Inc
Original Assignee
Aprogenex 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 Aprogenex Inc filed Critical Aprogenex Inc
Priority to AU69497/94A priority Critical patent/AU6949794A/en
Publication of WO1994026104A1 publication Critical patent/WO1994026104A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood

Definitions

  • a variety of fetal cell types platelets, trophoblasts, erythrocytes and leucocytes -- cross the placenta and circulate transiently within maternal blood
  • the present invention provides a method of separating a target cell population from a heterogeneous population of cells suspended in a liquid medium.
  • the method comprises repeatedly passing through the cell suspension at their gravitational settling rate therein particles having attached to the exterior thereof, directly or through linking groups, multiple target cell-specific binding species; and separating target cells bound to the particles from the heterogeneous cell suspension.
  • the method preferably further comprises the step of releasing the target cells from the particles.
  • the particularly heterogeneous cell suspension is preferably a suspension of maternal blood cells, or erythrocyte-poor fractions isolated therefrom, such as from a peripheral blood sample, and the target cells are preferably fetal cells.
  • the method is realized by selection of beads by size, density and shape so that the competing factors of (1) volume swept out by the bead per unit of time and (2) time of interaction between the bead and any one target cell that is encountered is optimized.
  • the first factor is emphasized with fast moving beads, the second with slow moving beads.
  • the method is particularly desirable when the target cells comprise a small fraction (less than 1:1000) of the total cells present and when the combination of bead density and size results in beads that are large enough easily to be separated from the cell suspension by collecting them on a cloth mesh that easily passes unadhered cells.
  • the invention provides a method of separating a target cell population from a heterogeneous cell suspension in a liquid medium.
  • the method comprises repeatedly passing through the cell suspension at their gravitational settling rate therein particles having attached to the exterior thereof, directly or through linking groups, multiple, target cell-specific binding partners; and separating target cells bound to the particles from the heterogeneous cell suspension.
  • the particles are passed through the suspension at a rate not greater than about 0.2 cm/sec.
  • the particles are passed through the suspension so as to achieve a peak surface shear rate in the range of 1-300 reciprocal seconds.
  • the particles can have a density greater or lesser than the suspension medium. It is the difference in density, used to achieve the desired peak shear rate, that is the important factor in the invention.
  • the particles have a density which preferably differs from that of the medium by not more than about 10%, most preferably by not more than about 5%.
  • the particles are essentially spherical (a typical diameter is about 50-150 micrometers, preferably about 100 micrometers) and can, optionally, have fibrous projections on their surfaces. Also, the particles can be connected by fibrous bridges.
  • the process of the invention comprises inverting at regular intervals a closed vessel containing the suspension and particles.
  • the closed vessel is repeatedly inverted at intervals equal to or shorter than the full- distance settling time of the particles in the cell suspension.
  • the vessel is inverted at substantially regular intervals for a period of up to about 24 hours. For example, for a cell suspension in a 10ml test tube, the tube is inverted about every 3 minutes for a duration of about 9 hours.
  • the invention makes it possible to recover a few thousand or less cells of a particular type (which are distinguished by characteristic molecule(s) on the surface of the cells to be recovered) from a heterogeneous cell suspension of up to several hundred million cells not possessing the characteristic molecule(s).
  • the recovery is effected by the use of a limited number of particles that are caused to move (search) slowly through the cell suspension.
  • a molecule capable of interacting reversibly with the molecule that characterizes the cells to be recovered is attached in multiple copies to each of the particles.
  • the size and velocity of searching of the particles is adjusted to optimize the rate of binding of the desired cells to the particles.
  • the particles are recovered with the cells attached.
  • the optimum dimensions and velocity of the particles are determined principally by specification of the particle shape and the "shear rate" of fluid at the surface of the particle during the cell capturing process.
  • Particle velocity is fixed by the choices of density (relative to that of the fluid in which the cells are suspended) and size of the particles.
  • the gentleness and biocompatibility of the process are used to allow long times of exposure, up to 24 hours. During this time the suspension is agitated (inverted) only often enough to keep the cells and particles in suspension, the important relative movement between particles and cells being imposed by gravity acting differentially on the particles.
  • the cells are released after the particles are separated from the original suspension and washed by exposing the particles to chemical or mechanical conditions which overcome the binding between the characteristic molecules on the cell surface and the complementary molecules on the particles.
  • the cells are released by simple collection of the particles on a cloth screen with openings of about 70 micrometers.
  • the particles are washed by applying drops of pH 2.8 citric acid solution, which detach the cells and allow the cells to fall into a volume of about 10 ml of cell culture medium.
  • the present invention teaches that the number of beads and the number of undesired attachments may be minimized with the number of desired attachments maximized by inducing slow movement of particles through the suspension over a period of time sufficient thoroughly to search the whole suspension volume, up to 24 hours if necessary.
  • the much greater surface area to which cells may be exposed in beds of particles is compensated for in this invention by a much longer time of exposure.
  • 10 ml of suspension is in the range of about 100-400 particles, preferably about
  • the only equipment required to maintain particle motion is a device which inverts a tube containing the suspension at a time interval less than or equal to the end-to-end settling time of the particles.
  • This device can be very simple and inexpensive.
  • the invention is particularly envisioned for use on the "buffy coat" fraction of maternal human blood to isolate the few fetal cells present in the donor's blood. These cells contain information concerning the fetus which is equivalent to that obtained from analysis of cells usually obtained by the riskier and more expensive process of amniocentesis. Many other diagnostic and therapeutic applications are envisioned, including isolation of cells belonging to a particular lymphocyte clone. Cells can be more completely and more easily recovered for propagation and reinfusion, which is advantageous in transplantation therapies now under active development in several medical centers. In biotechnology, mutant and phenotypically variant cells can be recovered from any population of cells capable of being brought into suspension.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un procédé d'isolement d'un petit nombre de cellules cibles d'une suspension de cellules hétérogènes dans un milieu liquide qui consiste à faire passer plusieurs fois des particules à l'extérieur desquelles sont fixées des espèces de liaison spécifiques à cellule cible dans la suspension de cellules hétérogènes à leur vitesse de sédimentation ou de montée par gravitation, à séparer les cellules cibles fixées sur les particules de la suspension de cellules hétérogènes. Ledit procédé peut également prévoir le détachement des cellules cibles des particules. La suspension de cellules hétérogènes est, de préférence, une suspension de cellules de sang maternel et les cellules cibles sont, de préférence, des cellules f÷tales.
PCT/US1994/005408 1993-05-14 1994-05-13 Procede d'isolement de cellules cibles Ceased WO1994026104A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU69497/94A AU6949794A (en) 1993-05-14 1994-05-13 Target cell isolation method

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US6200493A 1993-05-14 1993-05-14
US08/062,004 1993-05-14

Publications (1)

Publication Number Publication Date
WO1994026104A1 true WO1994026104A1 (fr) 1994-11-24

Family

ID=22039597

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/005408 Ceased WO1994026104A1 (fr) 1993-05-14 1994-05-13 Procede d'isolement de cellules cibles

Country Status (2)

Country Link
AU (1) AU6949794A (fr)
WO (1) WO1994026104A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5962237A (en) * 1996-04-05 1999-10-05 The Johns Hopkins University School Of Medicine Method of enriching rare cells
US6355174B1 (en) 1994-11-08 2002-03-12 Phoenix Medical Limited Method of separating foetal trophoblasts from maternal blood
US7439062B2 (en) 2004-12-23 2008-10-21 Biocept, Inc. Beads for capturing target cells from bodily fluid

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3709361A (en) * 1970-05-26 1973-01-09 Ontario Cancer Inst Device for separating living cells
US5004681A (en) * 1987-11-12 1991-04-02 Biocyte Corporation Preservation of fetal and neonatal hematopoietic stem and progenitor cells of the blood
US5215926A (en) * 1988-06-03 1993-06-01 Cellpro, Inc. Procedure for designing efficient affinity cell separation processes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3709361A (en) * 1970-05-26 1973-01-09 Ontario Cancer Inst Device for separating living cells
US5004681A (en) * 1987-11-12 1991-04-02 Biocyte Corporation Preservation of fetal and neonatal hematopoietic stem and progenitor cells of the blood
US5004681B1 (en) * 1987-11-12 2000-04-11 Biocyte Corp Preservation of fetal and neonatal hematopoietic stem and progenitor cells of the blood
US5215926A (en) * 1988-06-03 1993-06-01 Cellpro, Inc. Procedure for designing efficient affinity cell separation processes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CELLULAR IMMUNOLOGY, Volume 40, issued 1978, T. TIMONEN et al., "Human Natural Cell-Mediated Cytoxicity Against Fetal Fibroblasts", pages 69-78. *
JOURNAL OF IMMUNOLOGICAL METHODS, Volume 122, issued 1989, D. PILLING et al., "The Kinetics of Interaction Between Lymphocytes and Magnetic Polymer Particles", pages 235-241. *
JOURNAL OF IMMUNOLOGICAL METHODS, Volume 142, Number 1, issued 1991, A.P. GEE et al., "Effects of Target Antigen Density on the Efficacy of Immunomagnetic Cell Separation", pages 127-136. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6355174B1 (en) 1994-11-08 2002-03-12 Phoenix Medical Limited Method of separating foetal trophoblasts from maternal blood
US5962237A (en) * 1996-04-05 1999-10-05 The Johns Hopkins University School Of Medicine Method of enriching rare cells
US7439062B2 (en) 2004-12-23 2008-10-21 Biocept, Inc. Beads for capturing target cells from bodily fluid

Also Published As

Publication number Publication date
AU6949794A (en) 1994-12-12

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