Language selection

Search

Patent 2405881 Summary

Third-party information liability

Some of the information on this Web page has been provided by external sources. The Government of Canada is not responsible for the accuracy, reliability or currency of the information supplied by external sources. Users wishing to rely upon this information should consult directly with the source of the information. Content provided by external sources is not subject to official languages, privacy and accessibility requirements.

Claims and Abstract availability

Any discrepancies in the text and image of the Claims and Abstract are due to differing posting times. Text of the Claims and Abstract are posted:

  • At the time the application is open to public inspection;
  • At the time of issue of the patent (grant).
(12) Patent: (11) CA 2405881
(54) English Title: METHOD FOR SEPARATING CELLS
(54) French Title: METHODE DE SEPARATION DE CELLULES
Status: Granted
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 5/07 (2010.01)
  • C12M 1/26 (2006.01)
  • C12N 1/00 (2006.01)
  • C12N 5/00 (2006.01)
  • C12Q 1/24 (2006.01)
(72) Inventors :
  • WOODSIDE, STEVEN M. (Canada)
(73) Owners :
  • STEMCELL TECHNOLOGIES INC. (Canada)
(71) Applicants :
  • STEMCELL TECHNOLOGIES INC. (Canada)
(74) Agent: BERESKIN & PARR LLP/S.E.N.C.R.L.,S.R.L.
(74) Associate agent:
(45) Issued: 2011-11-15
(22) Filed Date: 2002-10-01
(41) Open to Public Inspection: 2003-04-01
Examination requested: 2007-09-07
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data:
Application No. Country/Territory Date
2,358,326 Canada 2001-10-01

Abstracts

English Abstract

The present invention is related to a method for separating a first population of cells from a second population of cells in a sample by discontinuous density gradient separation using dense particles to target the first population of cells and a density separation medium (DSM) that is at least about 0.001 g/cm3 higher than the density of the second population of cells.


French Abstract

La présente invention porte sur une méthode qui consiste à séparer une première population de cellules d'une seconde population de cellules d'un échantillon. Il s'agit d'effectuer une séparation à gradients de densités discontinue au moyen de particules denses pour cibler la première population de cellules, et d'un milieu de séparation de densités étant au moins environ 0,001 g/cm3 plus élevé que la densité de la seconde population de cellules.

Claims

Note: Claims are shown in the official language in which they were submitted.



30
WE CLAIM:

1. A method for separating a first population of cells from a second
population of cells in a sample comprising:
- linking dense particles to the first population of cells in the sample,
- layering the sample over a discontinuous density separation
medium (DSM) having a density at least about 0.001 g/cm3 greater
than the mean density of the second population of cells, and
- allowing the cells to settle,
wherein the particle-linked first population of cells will settle to below the

interface between the DSM and the sample and the second population of cells
will settle to the interface between the DSM and the sample

2. The method according to claim 1, further comprising recovering the
second population of cells from the interface between the discontinuous DSM
and the sample.

3 The method according to claim 1, further comprising recovering the first
population of cells from below the interface between the discontinuous DSM
and the sample

4. The method according to claim 3, further comprising removing the
dense particles from the first population of cells

The method according to any one of claims 1-4, wherein the settling is
accelerated by centrifugation

6. The method according to any one of claims 1-6, wherein the dense
particles are selected from the group consisting of red blood cells, silica
particles, metal particles, metal oxide particles, polymer particles and glass

particles.

7 The method according to claim 6, wherein the dense particles are


31
selected from the group consisting of red blood cells and silica particles

8 The method according to any one of claims 1-7, wherein the osmolarity
of the discontinuous DSM is approximately the same as the osmolarity of the
sample.

9. The method according to any one of claims 1-8, wherein the osmolarity
of the discontinuous DSM is about 270 to about 300 mOsm

10. The method according to any one of claims 1-9, wherein the first
population of cells are linked to the dense particles by drug-drug receptor,
antibody-antigen, hormone-hormone receptor, growth factor-growth factor
receptor, carbohydrate-lectin, nucleic acid sequence-complementary nucleic
acid sequence, enzyme-cofactor or enzyme-inhibitor binding

11. The method according to any one of claims 1-10, wherein the first
population of cells are defined by specific surface proteins and the dense
particles are linked to these cells by antibodies specific for the cell
surface
proteins

12. The method according to any one of claims 1-11, wherein the
discontinuous DSM has a density at least about 0 002 g/cm3 greater than the
mean density of the second population of cells

13. The method according to claim 12, wherein the discontinuous DSM has
a density at least about 0 004 g/cm3 greater than the mean density of the
second population of cells.

14. The method according to claim 2, wherein the second population of
cells is selected from the group consisting of T cells, B cells, basophils, NK

cells, dendritic cells, monocytes, macrophages, megakaryocytes, platelets,
eosinophils, neutrophils, hematopoietic stem cells, mesenchymal stem cells,
endothelial cells, epithelial cells, fibroblasts and tumour cells


32
15. The method according to claim 3, wherein the first population of cells is
selected from the group consisting of T cells, B cells, basophils, NK cells,
dendritic cells, monocytes, macrophages, megakaryocytes, platelets,
eosinophils, neutrophils, hematopoietic stem cells, mesenchymal stem cells,
endothelial cells, epithelial cells, fibroblasts and tumour cells

16. A kit for performing the method according to claim 1 comprising one or
more aliquots of a discontinuous DSM having a density at least about 0 001
g/cm3 higher than the mean density of the second population of cells, one or
more aliquots of cell-specific targeting agents and one or more aliquots of
dense particles.

17. The kit according to claim 16, wherein the dense particles are red
blood cells.

18. The kit according to claim 17, wherein the cell-specific targeting agents
are tetrameric antibody complex cocktails that link the first population of
cells
to the red blood cells.

19. The kit according to claim 16, wherein the dense particles are silica
particles.

20. The kit according to claim 16, wherein the cell-specific targeting agents
are tetrameric antibody complexes that link the first population of cells to
the
dense particles.

21 The kit according to claim 16, wherein the cell-specific targeting agent
is an antibody or antibody cocktail that binds the first population of cells
and
the dense particles bind to the first population of cells through interactions

with the antibody or antibodies in the cocktail.

22. A kit for performing the method according to claim 1 comprising one or


33
more aliquots of a discontinuous density separation medium having a density
at least about 0.001 g/cm3 higher than the mean density of the second
population of cells, and one or more aliquots of dense particles coated with
one or more cell-specific binding agents that bind the first population of
cells
23. The kit according to claim 22, wherein the dense particles are red
blood cells.

24. The kit according to claim 22, wherein the dense particles are silica
particles.

25. The kit according to claim 24, wherein the dense particles are coated
with one or more antibodies that bind the first population of cells.

26. The kit according to any one of claims 16-24, further comprising printed
instructions.

27. A use of a discontinuous density separation medium (DSM), for
discontinuous density gradient separation of a first population of cells from
a
second population of cells in a sample, wherein dense particles are linked to
the first population of cells, and the discontinuous DSM has a density that is
at
least about 0.001 g/cm3 greater than the mean density of the second
population of cells which settle at an interface between the discontinuous
DSM and the sample.

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02405881 2002-10-01

-1 -

B&P File No. 7771-92
TITLE: METHOD FOR SEPARATING CELLS
FIELD OF THE INVENTION
The present invention relates to methods for separating cells. In
particular the invention relates to methods for separating cells using dense
particles and discontinuous density gradient centrifugation.
BACKGROUND OF THE INVENTION
In many applications it is desirable to enrich, or alternatively deplete,
certain cell populations in a biological sample. For example, the separation
of
specific cell types from peripheral blood, bone marrow, spleen, thymus and
fetal liver is key to research in the fields of haematology, immunology and
oncology, as well as diagnostics and therapy for certain malignancies and
immune disorders.
Most cell separation techniques require that the input sample be a
single cell suspension. For this reason, blood has historically been the most
common tissue used for cell separations. Purified populations of immune cells
such as T cells and antigen presenting cells are necessary for the study of
immune function and are used in immunotherapy. Investigation of cellular,
molecular and biochemical processes requires analysis of certain cell types in
isolation. Numerous techniques have been used to isolate T cell subsets, B
cells, basophils, NK cells and dendritic cells from blood for these
investigations.
More recently, enzymatic digestion methods have been developed to
dissociate tissues from solid organs into single cell suspensions, permitting
distinct cell types to be isolated. This is of particular benefit to the study
of
pluripotent stem cells and tissue-specific stem cells from adults. The rapidly
growing field of stem cell research is spurred by the potential of these cells
to
repair diseased or damaged tissues. Bone marrow (hematopoietic) stem cells
were the first adult stem cells to be purified and used clinically and the
therapeutic potential of hematopoietic stem cells is now well documented.
Transplantation of hematopoietic cells from peripheral blood and/or bone
marrow is increasingly used in combination with high-dose chemo- and/or


CA 02405881 2002-10-01

-2-
radiotherapy for the treatment of a variety of disorders including malignant,
nonmalignant and genetic disorders. Very few cells in such transplants are
capable of long-term hematopoietic reconstitution, and thus there is a strong
stimulus to develop techniques for purification of hematopoietic stem cells.
Furthermore, serious complications and indeed the success of a transplant
procedure is to a large degree dependent on the effectiveness of the
procedures that are used for the removal of cells in the transplant that pose
a
risk to the transplant recipient. Such cells include T lymphocytes that are
responsible for graft versus host disease (GVHD) in allogenic grafts, and
tumor cells in autologous transplants that may cause recurrence of the
malignant growth. It is also important to debulk the graft by removing
unnecessary cells and thus reducing the volume of cyropreservant to be
infused.
In certain instances it is desirable to remove or deplete tumor cells from
a biological sample, for example in bone marrow transplants. Epithelial
cancers of the bronchi, mammary ducts and the gastrointestinal and
urogenital tracts represent a major group of solid tumors seen today.
Micrometastatic tumor cell migration is thought to be an important prognostic
factor for patients with epithelial cancer (Braun et al., 2000; Vaughan et
al.,
1990). The ability to detect such metastatic cells is limited by the
effectiveness of tissue or fluid sampling and the sensitivity of tumor
detection
methods. A technique to enrich circulating epithelial tumor cells in blood
samples would increase the ability to detect metastatic disease and facilitate
the study of such rare cells to determine the biological changes which enable
spread of the disease.
Hematopoietic cells and immune cells have been separated on the
basis of physical characteristics such as density and on the basis of
susceptibility to certain pharmacological agents which kill cycling cells. The
advent of monoclonal antibodies against cell surface antigens has greatly
expanded the potential to distinguish and separate distinct cell types. There
are two basic conceptual approaches to separating cell populations from
blood and related cell suspensions using monoclonal antibodies. They differ


CA 02405881 2002-10-01

-3-
in whether it is the desired or undesired cells which are
distinguished/labeled
with the antibody(s).
In positive selection techniques the desired cells are labeled with
antibodies and removed from the remaining unlabeled/unwanted cells. In
negative selection, the unwanted cells are labeled and removed.
Antibody/complement treatment and the use of immunotoxins are negative
selection techniques, but FACS sorting and most batch-wise
immunoadsorption techniques can be adapted to both positive and negative
selection. In immunoadsorption techniques, cells are selected with
monoclonal antibodies and preferentially bound to a surface which can be
removed from the remainder of the cells e.g. column of beads, flask, magnetic
particles. Immunoadsorption techniques have won favour clinically and in
research because they maintain the high specificity of cell targeting with
monoclonal antibodies, but unlike FACSorting, they can be scaled up to
directly process the large numbers of cells in a clinical harvest and they
avoid
the dangers of using cytotoxic reagents such as immunotoxins and
complement. They do however, require the use of a "device" or cell
separation surface such as a column of beads, panning flask or magnet.
Current techniques for the isolation of hematopoietic stem cells,
immune cells and circulating epithelial tumor cells all involve an initial
step to
remove red cells prior to antibody mediated adherence to a device or
artificial
particle (Firat et al., 1988; de Wynter et al., 1975; Shpall et al., 1994;
Thomas
et al., 1994; Miltenyi Biotec Inc., Gladbach, Germany). In the case of
positive
selection there is yet another step; removal of the cells from the device or
particle. These multiple steps require time and incur cell loss.
Discontinuous density gradient centrifugation is commonly used to
isolate peripheral blood mononuclear cells from granulocytes and
erythrocytes. Ficoll-Paque (Amersham Pharmacia Biotech AB, Uppsala
Sweden) is one of the most popular density separation solutions used for this
application. In a Ficoll density separation whole blood is layered over
Ficoll,
and then centrifuged. The erythrocytes, granulocytes and approximately 50%
of the mononuclear cells settle to the cell pellet while the remaining 50% of


CA 02405881 2002-10-01

-4-
the mononuclear cells settle to the Ficoll plasma interface. The success of
this technique relies on the difference in density between mononuclear cells
and granulocytes/erythrocytes as well as the choice of the density separation
medium (DSM). During centrifugation, cells that are more dense than the
DSM settle through the DSM forming a pellet at the bottom of the tube, while
cells that are less dense than the DSM collect at the interface between the
DSM and the cell suspension medium (e.g. plasma in the case of peripheral
blood, cell culture medium in the case of cultured cells or dissociated tissue
cells). Multiple layers of DSM having different densities can be used to
divide
the cells into multiple fractions. The same effect can be achieved by
centrifuging cells in a medium with a continuous density gradient and then
collecting the cells from different positions in the gradient. Sedimentation
rate
can also be used to separate cells of different density, but the separation
time
is influenced not only by density but also the viscosity of the suspension and
the cell size.
All density separation techniques have the same basic limitation; they
can not separate subpopulations of cells with overlapping density
distributions
such as human lymphocyte subsets. Simple density separation techniques
do not offer the high cell specificity offered by antibody-mediated
techniques.
To address this, dense particles have been targeted to cells using monoclonal
antibodies with affinity to cells surface antigens and used in discontinuous
or
continuous density gradient centrifugation to separate cell populations with
similar densities (Bildirici and Rickwood, 2001; Bildirici and Rickwood, 2000;
Patel and Rickwood, 1995; Patel et al. 1993; US patent 5,840,502; and
StemCell Technologies, Supplement to 1999/2000 Catalogue). There are two
advantages of using a discontinuous density gradient rather than a continuous
density gradient in dense particle mediated cell separation. Discontinuous
gradients are easier to prepare and offer a visible boundary (interface) where
cells not bound to particles selectively collect.
Several patents (US 5,840,502, US 5,648,223, US 5,646,004 and US
5,474,687) describe the use of dense particles for negative selection by
selectively targeting and pelleting undesired cell types using discontinuous


CA 02405881 2002-10-01

-5-
density gradient separations. These patents state that the optimum density of
the DSM for dense particle separation is within 0.0005 to 0.0002 g/cm3 of
the density of the desired cell population.
There are no documented techniques for positive selection of cells
using dense particles and discontinuous density gradients. In positive
selection the desired cells are targeted with antibodies and dense particles
and pelleted during separation.
SUMMARY OF THE INVENTION
The invention relates to the use of dense particles and discontinuous
density gradients to separate populations of cells from a mixed cell
suspension. It has been found that the use of dense particles to target a
first
population of cells and a density separation medium (DSM) with a density at
least 0.001 g/cm3 higher than the density of a second population of cells not
targeted by the dense particles, offers more effective cell separation through
increased recovery of the non-targeted population of cells at the interface
between the DSM and the sample suspension without affecting the pelleting
of the targeted cells. This is contrary to current practice and the teachings
of
US Patent Nos. 5,840,502, US 5,648,223, US 5,646,004 and US 5,474,687,
which teach that the DSM should have a density preferably within 0.0005
g/cm3 and more preferably within t 0.0002 g/cm3 of the desired cell density.
In these patents, the desired cells are not targeted by the dense particles.
Accordingly, the present invention provides a method for separating a
first population of cells from a second population of cells in a sample
comprising:
- linking dense particles to the first population of cells in the sample;
- layering the sample over a density separation medium (DSM)
having a density at least about 0.001 g/cm3 greater than the mean
density of the second population of cells; and
- allowing the cells to settle,
wherein the particle-linked first population of cells will settle to below the
interface between the DSM and the sample and the second population of cells
will settle to the interface between the DSM and the sample.


CA 02405881 2002-10-01

-6-
In the method of the invention, the first population of cells (i.e. those
linked to the dense particles) will settle out below the interface between the
DSM and the sample and the second population of cells will settle at the
interface between the DSM and the sample. Each population of cells may be
thus isolated or recovered.
The method of the invention may be used to positively or negatively
select a population of desired cells. For negative selection, the desired
cells
will be the second populations of cells and are accordingly recovered from the
interface between the DSM and the sample. For positive selection, the
desired cells will be the first population of cells, which are linked to the
dense
particles, and thus are recovered from the area below the interface between
the DSM and the sample. When positive selection techniques are used, it is
typically desired to remove the dense particles from the desired cells using
enzymatic, chemical or physical means.
In embodiments of the invention, the settling of the cells is accelerated
by centrifugation.
The invention includes all uses of the above-described methods as well
as kits to perform the methods of the invention.
Other features and advantages of the present invention will become
apparent from the following detailed description. It should be understood,
however, that the detailed description and the specific examples while
indicating preferred embodiments of the invention are given by way of
illustration only, since various changes and modifications within the spirit
and
scope of the invention will become apparent to those skilled in the art from
this detailed description.
DETAILED DESCRIPTION OF THE INVENTION
(i) Methods of the Invention
As hereinbefore stated, it has been found that the use of dense
particles to target a first population of cells and a density separation
medium
(DSM) with a density at least 0.001 g/cm3 higher than the mean density of the
a second population of cells not targeted by the dense particles, offers more
effective cell separation than separations according to current practice.


CA 02405881 2002-10-01

-7-
Accordingly, the present invention relates to a method for separating a
first population of cells from a second population of cells in a sample
comprising:
- linking dense particles to the first population of cells in the sample;
- layering the sample over a density separation medium (DSM)
having a density at least about 0.001 g/cm3 greater than the mean
density of the second population of cells; and
- allowing the cells to settle,
wherein the particle-linked first population of cells will settle to below the
interface between the DSM and the sample and the second population of cells
will settle to the interface between the DSM and the sample.
As used herein, the "first population of cells" (i.e. those linked to the
dense particles) are the cells which settle out below the interface between
the
DSM and the sample and the "second population of cells" are those cells
which settle at the interface between the DSM and the sample. As used
herein, the term "below the interface between the DSM and the sample"
typically means in a pellet at the bottom of the container. Due to this
difference in location, each population of cells may be isolated or recovered.
Accordingly, the present invention further comprises the step of isolating the
first population of cells from below the interface between the DSM and the
sample and/or isolating the second population of cells from the interface
between the DSM and the sample. When the density of the DSM is at least
about 0.001 g/cm3 greater than the mean density of the second population of
cells, more effective cell separations are achieved compared to separations
using currently practiced methods.
As used herein, the term "below the interface between the DSM and
the sample" typically means in a pellet at the bottom of the container.
The term "desired cells" as used herein refers to a population of cells
that one wishes to isolate from a sample. The population of desired cells may
contain one or more cells types.
The term "undesired cells" as used herein refers to a population of cells
that one wishes to remove from a sample. The population of undesired cells


CA 02405881 2002-10-01

-8-
may contain one or more cell types.
In embodiments of the invention, the cells are settled using gravity or
centrifugation. In preferred embodiments, the settling of the cells is
accelerated using centrifugation.
When the desired cells are the second population of cells, a negative
selection protocol is employed. In this instance, the dense particles are
targeted to the first population of cells, i.e. undesired cells, which settle
to an
area below the interface between the DSM and the sample. When
centrifugation is employed to accelerate settling, the first population of
cells
will settle more quickly to the bottom of the sample container to form a
pellet.
The second population of cells, or desired cells, may be recovered from the
interface between the DSM and the sample. Accordingly, the method of the
present invention further comprises the step of recovering the second
population of cells from the interface between the DSM and the sample.
When the desired cells are the first population of cells, a positive
selection protocol is employed. In this instance, the dense particles are
targeted to the desired cells, which settle to an area below the interface
between the DSM and the sample. When centrifugation is employed to
accelerate settling, the desired cells will settle more quickly to the bottom
of
the container to form a pellet. When positive selection is employed, it is
desirable to remove the dense particles from the cells. Accordingly, the
method of the present invention further comprises the step of recovering the
first population of cells from an area below the interface between the DSM
and the sample and, optionally, removing the dense particles from the first
population of cells. Removal of the dense particles from the first population
of
cells may be carried out using enzymatic, chemical or physical methods well
known in the art. For example, using proteolytic enzymes such, as papain.
In a preferred embodiment of the invention, the first population of cells
is the undesired cells and the second population of cells are the desired
cell,
or a negative selection protocol is used to separate desired cells from
undesired cells.


CA 02405881 2002-10-01

-9-
The sample can be any cell suspension from which one wishes to
separate desired cells. As such, the sample will generally contain a mixture
of
desired cells and undesired cells suspended typically as single cells in a
liquid
medium. The sample can be obtained from both in vivo and in vitro sources.
Examples of sources include, but are not limited to, peripheral blood, bone
marrow, spleen, thymus and fetal liver. Specific cell types that may be
isolated using the method of the invention include, but are not limited to, T
cells, B cells, basophils, NK cells, dendritic cells, monocytes, macrophages,
megakaryocytes, platelets, eosinophils, neutrophils, hematopoietic stem cells,
mesenchymal stem cells, endothelial cells, epithelial cells, fibroblasts and
tumour cells.
A variety of commercially available materials may be used as the DSM
in the method of the invention, including, but not limited to Ficoll-Paque ;
Lymphoprep ; any sugar solution, e.g. sucrose; dextran; any protein solution,
e.g. bovine serum albumin (BSA); iodinated low molecular weight compounds
such as Metrizamide and heavy salts, e.g. cesium chloride. In an
embodiment of the invention, the density separation medium (DSM) is
prepared by mixing hetastarch, iodixanol and water in different proportions
such that the desired density and osmolarity is obtained.
The density of the DSM should be at least about 0.001 g/cm3,
preferably about 0.002 g/cm3, more preferably about 0.004 g/cm3, higher, than
the mean density of the density of the second population of cells (i.e. the
cells
that are not targeted by the dense particles). As used herein, the term "mean
density of the second population of cells" refers to the density of the second
population of cells as determined using density gradient centrifugation with a
series of DSM having different densities and without dense particles (for
example, see Example 6 herein).
Mammalian cells and cells from most multi-cellular organisms have
semi-permeable membranes and are therefore sensitive to changes in the
osmolarity of their environment. Cell density will increase with increasing
osmolarity of the surrounding solution. The mean density of some human
blood cell types is given in Table 1 for solutions iso-osmolar to plasma (270-


CA 02405881 2002-10-01

_10-
290 mOsm) and for solutions hyper-osmolar to plasma (300-310 mOsm).
This table shows that the density of the cells is strongly dependent on
solution
osmolarity. Thus mean cell density of a given population is defined in the
context of the solution osmolarity. In discontinuous density gradient
separation, the mean cell density of a given population of cells is said to be
equal to the density of the DSM if the number of cells in the given population
recovered at the interface is approximately the same as the number
recovered in the pellet (or if 50% of the initially present second population
of
cells are recovered at the interface when recovery in the pellet cannot be
reliably determined).
The osmolarity of the DSM is preferably approximately the same as the
osmolarity of the sample (i.e the DSM is approximately iso-molar to the
sample). The term "approximately the same" means that the osmolarity of the
DSM is in the range of about plus or minus 10% of the osmolarity of the
sample. Most preferably, the osmolarity of the DSM is in the range of about
270 to about 300 mOsm.
The density of the DSM should also be lower than the effective density
of the first population of cells when linked to dense particles such that most
of
the particle-linked first population of cells settle to the pellet during
density
gradient separation.
The effective density of the particle-bound cells will depend on the size
and density of the cells and the size, density and number of particles linked
to
the cells. For any cell linked to dense particles, the effective density can
be
defined as the product of the cell volume (Vc) and cell density (pc), plus the
product of the total volume of the particles (Vp) linked to the cell and the
density of the particles (pp), all divided by the total volume of the cell and
the
particles linked to the cell:

Vcpc + Vppp
Vc+Vp


CA 02405881 2010-10-04

-11 -

For example, a cell with a diameter of 10 pm and a density of 1.0 g/cm3
linked to one particle with a diameter of 10 pm and a density of 2.0 g/cm3
would have an effective density of 1.5 g/cm3. The same cell linked to 9
particles with a diameter of 10 pm and a density of 2.0 g/cm3 would have an
effective density of 1.9 g/cm3.
The methods of the invention may be carried out in any suitable
container, for example a centrifuge tube or syringe.
The method of the invention can be used to separate cells into multiple
fractions by using multiple layers of DSM each having a density that is at
least
about 0.001 g/cm3 greater than the mean density of the population of cells
that one wishes to have settle at the interface between that DSM and the
sample. Accordingly, in a further embodiment of the present invention there is
provided a use, for discontinuous density gradient separation of one or more
populations of cells from a sample, of a DSM having a density that is at least
about 0.001 g/cm3 greater than the mean density of the population of cells
that is to settle at an interface between the DSM and the sample.
The use of particles to adjust the density of the undesired or desired
cells during density gradient cell separations is described in US 5,840,502,
In embodiments of the present invention, the first population of cells
may be linked to dense particles through various types of binding including,
but not limited to, drug-drug receptor, antibody-antigen, hormone-hormone
receptor, growth factor-growth factor receptor, carbohydrate-lectin, nucleic
acid sequence-complementary nucleic acid sequence, enzyme-cofactor or
enzyme-inhibitor binding. Preferably, the cells to be linked to the dense
particles are defined by specific surface proteins and the dense particles are
linked to these cells by antibodies specific for the cell surface proteins.
In one embodiment of the invention, the antibodies specific for cell
surface proteins on the cells to be linked to the dense particles are in an
antibody composition or cocktail. Examples of antibody compositions or
cocktails which may be used to target a range of cell types are described US
patent No. 6,448,075,


CA 02405881 2010-10-04

-12-
Examples of antibody compositions or cocktails which may be
used to target hematopoietic stem cells and progenitor cells are described in
US patent Nos. 5,877,299, 6,117,985 and 6,307,575.

In further embodiments of the present invention, the dense particles
serve to increase the effective density of the first population of cells and
may
be, for example, selected from the group consisting of red blood cells, silica
particles, metal particles, metal oxide particles, polymer particles, glass
particles. Red blood cells may be linked to the first population of cells
using
tetrameric antibody complexes as described in Example 2 herein. Other
particles may be linked to the first population of cells by coating the
particles
with, for example, antibodies or dextran.
Kits
Density separation media, cell specific targeting agents and/or dense
particles for the separation of specific cell types may be prepared and
packaged in convenient kits, packaged into suitable containers. Accordingly,
the present invention relates to a kit for separating a first population of
cells
from a second population of cells in a sample comprising one or more aliquots
of a density separation medium having a density at least about 0.001 g/cm3
higher than the mean density of the second population of cells, one or more
aliquots of cell-specific targeting agents to target dense particles to the
first
population of cells and one or more aliquots of dense particles.
In an embodiment of the invention, applications that use red blood cells
as dense particles to separate a first population of cells from a second
population of cells from whole peripheral blood, as in Examples 2 and 3, the
cell specific targeting reagents may include one or more aliquots of
tetrameric
antibody complex cocktails that link the first population of cells to the red
blood cells.
In a further embodiment of the invention, a kit for separating a first
population of cells from a second population of cells in a sample consisting
more generally of nucleated cells, may comprise one or more aliquots of a
density separation medium having a density at least about 0.001 g/cm3 higher


CA 02405881 2002-10-01

-13-
than the mean density of the second population of cells, and one or more
aliquots of cell targeting reagents. In embodiments of the invention the cell
targeting reagents are selected from dense particles coated with one or more
cell-specific binding agents that target the first population of cells, for
example, antibody coated dense particles that target the first population of
cells, or one or more aliquots of a cell-targeting reagent, for example an
antibody or antibody cocktail, that binds to the first population of cells and
one
or more aliquots of dense particles that then bind to those cells through
interactions with the cell targeting reagent. In specific embodiments of the
invention, the cell-targeting reagents are tetrameric antibody complexes.
With particular regard to density separation media packaged in "kit"
form, it is preferred that aliquots of the media be packaged in separate
containers, with each container including a sufficient quantity of reagent for
at
least one assay to be conducted. A preferred kit is typically provided as an
enclosure (package) comprising one or more containers for the within-
described reagents.
Printed instructions providing guidance in the use of the packaged
reagent(s) may also be included, in various preferred embodiments. The term
"instructions" or "instructions for use" typically includes a tangible
expression
describing the reagent concentration or at least one assay method parameter,
such as the relative amounts of reagent and sample to be mixed,
maintenance time periods for reagent/sample mixtures, temperature, buffer
conditions, and the like.
The following non-limiting examples are illustrative of the present
invention:
EXAMPLES
Example I - Preparation of Density Separation Media (DSM)
Density separation media (DSM) were prepared at different densities
by mixing hetastarch, iodixanol and water in different proportions such that
the
desired density was obtained and the osmolarity was between 270 and 300
mOsm. The hetastarch and iodixanol components of this DSM serve the


CA 02405881 2010-10-04

-14-
same function as the polysaccharide and metrizoate components respectively
in both Ficoll-Paque and Lymphoprep .
Example 2 - Method to Negatively Select Cells from Whole Human
Peripheral Blood Using the Method of the Invention with Red Blood
Cells as Dense Particles
Preparation of Tetramers
In order to prepare a tetrameric antibody complex for use in the method
of the present invention, the following protocol may be used: (a) take 1 mg of
antibody specific for cells to be bound for red blood cells (e.g. anti-CD2,
CD3,
CD4, CD8, CD14, CD16, CD19 etc.); (b) add 3 mg anti-Glycophorin A
antibody (against red blood cells); mix well (c) then add 4.0 mg of P9
antibody
or 2.72 mg of the P9 F(ab')2 antibody fragment. Incubate overnight at 37 C.
The P9 antibody binds the Fc portion of the antibodies added in steps (a) and
(b) resulting in a tetrameric antibody complex. For more information on the
preparation of tetramers see U.S. Patent No. 4,868,109 to Lansdorp,
Tetrameric antibody complexes
incorporating different antibodies to antigens expressed on nucleated cells
are
prepared separately and then mixed.
The antibody compositions are made by combining various tetrameric
antibody complexes depending on which cells one wishes to pellet during
density gradient separation. The concentration of the various tetrameric
antibody complexes varies: typically antibodies to antigens expressed on
nucleated cells are at 10-30 ;tg/mL in tetrameric complexes. The composition
is then diluted 1/10 into the cells so the final concentrations of each anti
nucleated cell antibody in the cell suspensions is 1.0-3.0 g/mL.
Separation method
A negative selection protocol for separating cells from whole peripheral
blood is set out below.
1. Add 100 L antibody composition per mL of whole peripheral blood.
2. Incubate 20 minutes at room temperature.
3. Dilute sample with an equal volume of phosphate buffered saline (PBS)
+ 2% fetal calf serum (FCS) and mix gently.


CA 02405881 2002-10-01

-15-
4. Layer the diluted sample on top of the DSM.
5. Centrifuge for 20 minutes at 1200 x g, room temperature, with the
brake off.
6. Remove the desired cells from the DSM:plasma interface.
7. Wash desired cells with 5-10x volume of PBS + 2% FCS.
Example 3 - Method to Positively Select Cells from Whole Human
Peripheral Blood Using the Method of the Invention with Red Blood
Cells as Dense Particles
Preparation of Tetramers
Tetrameric antibody complexes were prepared as described in
Example 2. The antibody compositions are one or more types of tetrameric
antibody complexes depending on which cells one wishes to select.
Separation method
A positive selection protocol for separating cells from peripheral blood
is set out below.
1. Add 100 L antibody composition per mL of peripheral blood.
2. Incubate 20 minutes at room temperature.
3. Dilute sample with an equal volume of phosphate buffered saline (PBS)
+ 2% fetal calf serum (FCS) and mix gently.
4. Layer the diluted sample on top of the DSM.
5. Centrifuge for 20 minutes at 1200 x g, room temperature, with the
brake off.
6. Remove the desired cells from the pellet.
7. Wash desired cells with 5-1 Ox volume of PBS + 2% FCS.
8. Lyse red blood cells by adding 10x volume of lysis buffer such as
ammonium chloride, incubate 5-10 min, spin down and resuspend in
PBS + 2% FCS
Example 4 -Method to Negatively Select Cells from Human Peripheral
Blood Mononuclear Cell Suspensions Using the Method of the Invention
with Silica Particles as Dense Particles


CA 02405881 2002-10-01

-16-
Preparation of Tetramers
In order to prepare a tetrameric antibody complex for use in the method
of the present invention, the protocol describe in Example 2 may be used with
the following changes: (a) take 1 mg of antibody specific for cells to be
pelleted (e.g. anti-CD2, CD3, CD4, CD8, CD14, CD16, CD19 etc.); (b) add 2
mg anti-Dextran antibody; mix well (c) then add 3.0 mg of P9 antibody or 2.04
mg of the P9 F(ab')2 antibody fragment.
The antibody compositions are made by combining various tetrameric
antibody complexes depending on which cells one wishes to pellet during
density gradient separation.
Preparation of Mononuclear Cell Suspension
Previously frozen mononuclear cells obtained by Ficoll-Paque density
gradient separation were thawed and washed once with a 1 Ox volume of PBS
+ 2% FCS. The cells were resuspended at 5x10' cells/ mL.
Separation method
A negative selection protocol for density gradient separation from
human peripheral blood mononuclear cells using silica particles is set out
below. The silica particles were coated with dextran.
1. Add dextran-coated silica particles to cell suspension and mix.
2. Add 100 L antibody composition per mL of cell suspension and mix.
3. Incubate 20 minutes at room temperature.
4. Dilute sample with an equal volume of phosphate buffered saline (PBS)
+ 2% fetal calf serum (FCS) and mix gently.
5. Layer the diluted sample on top of the DSM.
6. Centrifuge for 20 minutes at 1200 x g, room temperature, with the
brake off.
7. Remove the desired cells from the DSM: buffered saline interface
8. Wash desired cells with 5-10x volume of PBS + 2% FCS.
Example 5 -Method to Negatively Select Cells from Mouse Spleen Cell
or Bone Marrow Cell Suspensions Using the Method of the Invention
with Silica Particles as Dense Particles


CA 02405881 2002-10-01

-17.
A protocol for negative selection of cells from mouse spleen or bone
marrow is set out below.
1. Obtain single cell suspensions from murine spleen or bone marrow
using standard procedures
2. Add rat anti-mouse antibodies that bind to specific cell surface proteins
on unwanted cells at a final concentration of between 1.0 and 3.0
g/mL. For example to enrich for murine CD4+ cells, add antibodies
targeting CD11b, CD45R, CD8, erythroid cells (TER119) and myeloid
differentiation antigen (Gr-1).
3. Incubate for at least 5 min
4. Wash cells then incubate with silica particles that are coated with goat-
anti-mouse antibodies.
5. Layer the cell-particle mixture over DSM
6. Centrifuge for 20 min at 1200 x g with no brake
7. Remove desired cells from the DSM: buffered saline interface.
8. Wash desired cells with 5-10x volume of PBS +2% FCS.
Example 6 - Determination of Cell Densities
Three (3) mL of various DSM were aliquoted in separate 15 mL conical
polypropylene tubes. The DSM and their respective density and osmolarity
were Ficoll-Paque Plus from Pharmacia (1.077, g/cm3, 300-310 mOsm) and
three density separation media (DSM) prepared as described in Example 1
with densities of 1.077, 1.081 and 1.085 g/cm3 and Osm=290 mOsm. Single
cell suspensions were obtained from mouse spleens using standard
procedures. A total of 2 x 107 cells was diluted in 2 mL of the appropriate
buffer. The diluted cell suspensions were layered on top of each of the DSM.
The samples were centrifuged in a swinging bucket centrifuge at 1200 x g for
10 minutes at room temperature with the centrifuge brake off. The original
spleen cell suspension, light density cells (collected from the buffer:DSM
interface) and pellet cells were analyzed by flow cytometry.
The results in Table 2 show that the recovery of lymphocytes and
granulocytes increases with increasing density of the DSM up to 1.085 g/cm3.
The recovery with Ficoll-Paque is lower than with the DSM prepared with the


CA 02405881 2002-10-01

_18-
same density likely because it has a lower osmolarity than Ficoll-Paque (see
Table 1). A DSM density of 1.081 g/cm3 is ideal for depleting RBC while
maintaining a high lymphocyte recovery according to these data. The data in
Table 2 also show that the average lymphocyte and granulocyte density is
between 1.077 and 1.081 g/cm3. According to the prior art a DSM with a
density in this range is preferred for dense particle assisted density
separations. Further examples will show that the preferred DSM density is
higher than the average lymphocyte or granulocyte density. Although the data
in Table 2 relate specifically to mouse spleen cells, this type of analysis
can
be applied to any cell population to determine the recovery and purity as a
function of DSM density.
Example 7 -Effect of DSM Density on the Negative Selection of Murine
Spleen and Bone Marrow Cells Using Silica Particles and Discontinuous
Density Gradient Separation
CD4+ cells were enriched from murine spleen suspensions following
the method of Example 5 using a cocktail of antibodies targeting CD11b,
CD45R, CD8, erythroid cells (TER119) and myeloid differentiation antigen
(Gr-1). CD8+ cells were similarly enriched using a cocktail of antibodies
targeting CD11b, CD45R, CD4, erythroid cells (TER119) and myeloid
differentiation antigen. Murine progenitors, defined as Sca1+ / lineage
negative (CD3, CD11b, CD45R and Gr-1 negative), were enriched from
murine bone marrow using a cocktail of antibodies targeting CD5, CD11 b,
CD45R, erythroid cells (using TER antibody), myeloid differentiation antigen
(Gr-1) and neutrophiis (using 7-4 antibody).
The results in Table 3 show that recovery of the desired cells increases
with increasing density of the DSM and that the range of useful densities is
well above 1.081 g/cm3 which was shown in Example 6 to be the upper limit
of the density of the desired lymphocytes. Thus, it is advantageous to use a
DSM with a higher density than the density of the desired cell for murine cell
separations.


CA 02405881 2002-10-01

-19-
Example 8 - Effect of DSM Density on Negative Selection of Human
Peripheral Blood Cells using Red Blood Cells as Dense Particles and
Discontinuous Density Gradient Separation
Human peripheral blood cells were separated using the method
described in Example 2. The T (CD3+) cell enrichment cocktail included
antibody complexes targeting CD16, CD19, CD36 and CD56. The CD4+ T
cell enrichment cocktail included antibody complexes targeting CD8, CD16,
CD19, CD36 and CD56. The CD8+ T cell enrichment cocktail included
antibody complexes targeting CD4, CD16, CD19, CD36 and CD56. The B
(CD19+) cell enrichment cocktail included antibody complexes targeting CD2,
CD3, CD16, CD36 and CD56. The NK (CD56+) cell enrichment cocktail
included antibody complexes targeting CD3, CD4, CD19, CD36 and CD66b.
Table 4 shows data obtained from two different samples, where one
sample was incubated with a cocktail of antibody complexes to enrich T-cells
and the other was incubated with the B-cell enrichment cocktail. The samples
were then layered in 3 equal volume aliquots over each of Ficoll-Paque
(1.077 g/cm3, 300-310 mOsm) and two DSM prepared with densities of 1.081
and 1.085 g/cm3 (290 mOsm) and separated following Example 1. The
tabulated values are the average of the results for the n=3 separations for
each DSM. The results in Table 4 clearly show that the recovery of the
desired cells is improved with increased DSM density and that the purity of
the desired cell population obtained is not adversely affected up to a density
of 1.085 g/cm3.
The results summarized in Table 5 are from the separation of 3
samples where such sample was divided into 5 equal parts and incubated
with a cocktail of antibody complexes for the enrichment of the 5 desired cell
types listed following the method of Example 2. Each of these volumes was
then diluted, split into two equal volumes and layered over either Ficoll-
Paque (1.077 g/cm3, 300-310 mOsm) or a DSM prepared with a density of
1.081 g/cm3 (290 mOsm). Table 5 shows that the recovery of desired cells is
higher using a DSM with a higher density than Ficoll-Paque despite the
difference in osmolarity. The purity of the desired population is equivalent
or


CA 02405881 2002-10-01

-20-
higher using DSM. The optimum DSM density for the isolation of T, B and NK
cells from whole blood in conjunction with dense particle enrichment is thus
higher than that of Ficoll-Paque which is close to the density of MNC at 300-
310 mOsm (see Table 1) and is optimum for MNC isolation in the absence of
dense particle enrichment. This example shows that the preferred DSM
density for density separations of human lymphocyte subsets using red blood
cells as dense particles to deplete unwanted cells is substantially higher
than
the density of the desired lymphocytes.
Example 9 - Depletion of Undesired Human Peripheral Blood Cells
Using Dextran Coated Silica Particles and Discontinuous Density
Gradient Separation
Previously frozen mononuclear cells were incubated with anti-CD3:anti-
dextran bi-specific antibody complex (as described in Example 4) at 1.0
pg/mL anti-CD3 antibody for 20 min at room temperature, then washed to
remove excess tetramer. Dextran coated silica particles were then added at
2.5 g/mL, incubated for 20 minutes at room temperature and then diluted 4
fold and layered over either Ficoll-Paque (1.077 g/cm3, 300-310 mOsm) or a
DSM with a density of 1.081 g/cm3 (290 mOsm) in a centrifuge tube. The
tubes were centrifuged for 20 min at 1200x g with no brake and the cells at
the interface were recovered and washed 1x. The CD3+ cell log depletion
was determined by taking the log of the number of cells labeled with sheep
anti-mouse (SAM)-FITC+ in the start sample divided by the number in the final
sample. SAM antibodies bind to cells that have been targeted by murine
antibodies including the anti-CD3: anti-dextran bi-specific antibodies.
Table 6 shows that the log depletion of CD3+ cells was equivalent for
both DSM but that the recovery of CD3-negative cells was higher using DSM
(1.081 g/cm3, 290 mOsm) because more cells were buoyant than in Ficoll-
Paque . The density of Ficoll-Paque is close to the mean lymphocyte
density (see Table 1). This example shows that the preferred density of the
DSM for density separations using silica particles to deplete unwanted cells
is
higher than the lymphocyte density to provide improved recovery of CD3-
negative cells without affecting CD3+ cell depletion efficiency.


CA 02405881 2010-10-04

-21 -

Example 10 - Effect of DSM Density on Positive Selection of CD3+
Human Peripheral Blood Cells Using Red Blood Cells as Dense Particles
and Discontinuous Density Gradient Separation.
Three DSM were prepared following Example 1 with densities of 1.081,
1.085 and 1.090 g/cm3. Anti-CD3: aGIyA antibody complexes were prepared
as described in Example 2. Following the positive selection separation
method described in Example 5, four equal volume aliquots of blood were
incubated with the anti-CD3: aGIyA antibody complexes and layered over
each of Ficoll-Paque (1.077 g/cm3, 300-310 mOsm) and the three DSM with
densities of 1.081, 1.085 and 1.090 g/cm3 (290 mOsm) and separated by
centrifugation. The desired cells were recovered from the pellet and the red
blood cells were removed from the desired cells by lysis using an ammonium
chloride solution.
The results in Table 7 clearly show that the purity of the desired cells is
improved using a DSM with a density of 1.081 g/cm3 instead of Ficoll-Paque
(1.077 g/cm3, 300-310 mOsm). The undesired dense cells, such as
granulocytes, are more buoyant in the higher density DSM and do not settle to
the pellet. The recovery of desired cells generally decreases with increasing
DSM density. This is due to the greater buoyancy of the desired cells in DSM
of increasing density and the greater number of linked RBC necessary to
change the effective density of the cells such that they will pellet. This
example shows that the preferred DSM density for positive selection density
separations of human CD3+ cells using red blood cells as dense particles is
substantially higher than the density of Ficoll-Paque despite the difference
in
osmolarity and is higher than the density of the undesired cells.
While the present invention has been described with reference to what
are presently considered to be the preferred examples, it is to be understood
that the invention is not limited to the disclosed examples. To the contrary,
the
invention is intended to cover various modifications and equivalent
arrangements included within the spirit and scope of the appended claims.


CA 02405881 2010-10-04

22
Table 1 - Mean density of human peripheral blood cells in solutions
iso-osmolar to plasma and hyper-osmolar to plasma

Cell Density (g/cm) in:
Iso-osmolar solution Hyper-osmolar solution
Cell Type (270-290 mOsm) (300-310 mOsm) $
Monocytes 1.064 NA
Lymphocytes 1.072 1.077$
Neutrophils 1.078* NA
NA: not available
Nycomed Amersham "Centrifugation Techniques"
$ Ficoll-Paque Product information sheet


CA 02405881 2010-10-04

23
Table 2 Recovery of red blood cells (RBC), lymphocytes (Lymphs)
and granulocyte (Grans) populations in the interface and pellet after
density gradient centrifugation of murine spleen cells over density
media of various densities

interface Pellet
DSM Density RBC Lymphs Grans RBC Lymphs Granus
(g/cm3) % % recovery % recovery % % recovery % recovery
recovery recovery
1.077 * 0.2 20 18 76 20 79
1.077 $ 0.2 48 50 92 50 42
1.081 $ 0.3 68 38 > 100 1.4 2
1.085 $ 16 86 80 69 1 1.13
1.077 * < 0.1 5.3 10.4 > 100 83 96
1.077 $ 0.2 15 16 > 100 76 67
1.081 $ 0.3 101 >100 > 100 5.9 12
1.085 $ 3.6 >100 > 100 25 1.9 3.2
*Ficoll-Paque : 300-310 mOsm
$Other DSM: 290 mOsm


CA 02405881 2010-10-04

24
Table 3 - Purity and recovery of negatively selected murine cells using
GAR-coated silica particles and discontinuous density gradient
separation with DSM of different densities (290 mOsm).

Cell type DSM % Purity in % Purity in % Recovery in
enriched Density Start Enriched Enriched
g/CM3)
CD8+ cells
(n=2)
1.0950 9.4 88, 86 49,49
1.0900 9.4 90, 90 32, 29
Sca+, lin-
(n=2)
1.0900 1.6 10, 11 52, 39
1.0875 1.6 9.2, 9.4 47,41
1.0800 1.6 8.6, 8.5 33, 37
CD4+ cells
(n=2)
1.0900 21 84, 81 34,27
1.0875 21 88, 86 34,34
1.0800 21 84, 84 29, 30
CD4+ cells
(n=1)
1.0950 24 87 26
1.0900 24 89 23


CA 02405881 2010-10-04

Table 4 -Purity and recovery of human cells negatively selected
from whole blood using red blood cells as dense particles and
discontinuous density gradient separation with DSM of different
densities.

DSM Density /cm % purity desired cells % recovery desired cell
T cell (CD3+) enrichment
1.077 (Ficoll-PaqueR) 96 26
1.081 $ 96 44
1.085 $ 96 55
B cell (CD19+) enrichment
1.077 (Ficoll-Paque) 86 62
1.081 $ 88 96
1.085 $ 85 91
Ficoll-Paque : 300-310 mOsm
$Other DSM: 290 mOsm


CA 02405881 2010-10-04

26
Table 5 - Purity and recovery of human cells negatively selected
from whole blood using red blood cells as dense particles and
discontinuous density gradient separation with Ficoll-Paque (1.077
g/cm3, 300-310 mOsm) and DSM at 1.081 g/cm3 (290 mOsm).

Desired Cell Purity Recovery
Population Ficoll- DSM (1.081 Ficoll- DSM (1.081
Pa ue g/CM Pa ue /cm3)
T cell (CD3+) 98% 98% 61% 71%
CD4+ T cell 94% 94% 67% 78%
CD8+ T cell 85% 85% 42% 43%
B cell 84% 90% 72% 87%
(CD 19+)
NK cell 82% 85% 24% 27%
(CD56+)


CA 02405881 2010-10-04

27
Table 6 - Depletion of human CD3 positive T cells and recovery of
CD3 negative cells using silica particles and discontinuous density
gradient separation with Ficoll-Paque (1.077 g/cm3, 300-310 mOsm)
and DSM at 1.081 g/cm3 (290 mOsm).

DSM % CD3+ in % CD3+ in Log % Recovery
start depleted depletion CD3- cells
sample sample CD3+

DSM 1.081 g/cm 39 4.3 1.6 34
Ficoll-Pa ue 39 9.9 1.4 26
DSM 1.081 g/cm 39 4.0 1.7 1-41-
Ficoll-Paqueo 39 3.7 1.8 31


CA 02405881 2010-10-04

28
Table 7 - Purity and recovery of human CD3+ cells positively
selected from human blood using red blood cells as dense particles and
discontinuous density gradient separation.

DSM density (g/cm) % Purity % Recovery
1.077 (Ficoll-Paque) 80.2 42
1.081$ 93.6 51
1.085$ 89.0 43
1.090$ 83.9 41
Ficoll-Paque :300-310 mOsm
$Other DSM: 290 mOsm


CA 02405881 2010-10-04

29
FULL CITATIONS FOR REFERENCES REFERRED TO
IN THE SPECIFICATION

1. Braun et al., N. Engl. J. Med., 342:525:533.
2. deWynter, E.A. et al., 1975, Stem Cells, Vol. 13:524-532.
3. Firat et al., 1988, Bone Marrow Transplantation, Vol. 21:933-938.
4. Shpall, E.J., et al. 1994, J. of Clinical Oncology 12:28-36.
5. Thomas, T.E., 1994, Cancer Research, Therapy and Control 4(2): 119-
128.
6. Vaughan et al., 1990, Proc. Am. Soc. Clin. Oncol. 9:9.
7. Patel et al., 1995, Clinica Chimica Acta 240: 187-193.
8. Patel and Rickwood, 1995, J. Immunol. Meth. 184: 71-80.
9. Bildirici and Rickwood, 2000, J. Immunol. Meth. 240: 93-99.
10. Bildirici and Rickwood, 2001, J. Immunol. Meth. 252: 57-62.
11. Patel et al., 1993, J. Immunol. Meth., 163:241-251.
12. Van Vlasselaer US Patent 5,648,223
13. Van Vlasselaer US Patent 5,474,687
14. Van Vlasselaer US Patent 5,646,004
15. Van Vlasselaer US Patent 5,840,502
16. Coulter et al. US Patent 5,576,185
17. StemCell Technologies, 1999/2000 Catalogue supplement

Representative Drawing

Sorry, the representative drawing for patent document number 2405881 was not found.

Administrative Status

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Administrative Status , Maintenance Fee  and Payment History  should be consulted.

Administrative Status

Title Date
Forecasted Issue Date 2011-11-15
(22) Filed 2002-10-01
(41) Open to Public Inspection 2003-04-01
Examination Requested 2007-09-07
(45) Issued 2011-11-15

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $300.00 2002-10-01
Registration of a document - section 124 $100.00 2003-01-31
Maintenance Fee - Application - New Act 2 2004-10-01 $100.00 2004-09-09
Maintenance Fee - Application - New Act 3 2005-10-03 $100.00 2005-09-29
Maintenance Fee - Application - New Act 4 2006-10-02 $100.00 2006-09-27
Request for Examination $800.00 2007-09-07
Maintenance Fee - Application - New Act 5 2007-10-01 $200.00 2007-09-07
Maintenance Fee - Application - New Act 6 2008-10-01 $200.00 2008-09-29
Maintenance Fee - Application - New Act 7 2009-10-01 $200.00 2009-09-18
Maintenance Fee - Application - New Act 8 2010-10-01 $200.00 2010-08-24
Final Fee $300.00 2011-08-03
Maintenance Fee - Application - New Act 9 2011-10-03 $200.00 2011-09-12
Maintenance Fee - Patent - New Act 10 2012-10-01 $250.00 2012-09-06
Maintenance Fee - Patent - New Act 11 2013-10-01 $250.00 2013-09-25
Maintenance Fee - Patent - New Act 12 2014-10-01 $250.00 2014-09-19
Maintenance Fee - Patent - New Act 13 2015-10-01 $250.00 2015-09-21
Maintenance Fee - Patent - New Act 14 2016-10-03 $250.00 2016-09-14
Maintenance Fee - Patent - New Act 15 2017-10-02 $450.00 2017-09-12
Maintenance Fee - Patent - New Act 16 2018-10-01 $450.00 2018-09-17
Maintenance Fee - Patent - New Act 17 2019-10-01 $450.00 2019-09-23
Maintenance Fee - Patent - New Act 18 2020-10-01 $450.00 2020-09-11
Maintenance Fee - Patent - New Act 19 2021-10-01 $459.00 2021-09-17
Registration of a document - section 124 2022-06-30 $100.00 2022-06-30
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
STEMCELL TECHNOLOGIES INC.
Past Owners on Record
WOODSIDE, STEVEN M.
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

To view selected files, please enter reCAPTCHA code :



To view images, click a link in the Document Description column. To download the documents, select one or more checkboxes in the first column and then click the "Download Selected in PDF format (Zip Archive)" or the "Download Selected as Single PDF" button.

List of published and non-published patent-specific documents on the CPD .

If you have any difficulty accessing content, you can call the Client Service Centre at 1-866-997-1936 or send them an e-mail at CIPO Client Service Centre.


Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Maintenance Fee Payment 2020-09-11 1 33
Description 2002-10-01 30 1,338
Cover Page 2003-03-07 1 24
Change to the Method of Correspondence 2021-07-14 4 105
Maintenance Fee Payment 2021-09-17 1 33
Abstract 2002-10-01 1 13
Claims 2002-10-01 4 153
Description 2010-10-04 29 1,276
Claims 2010-10-04 4 144
Cover Page 2011-10-11 1 26
Correspondence 2002-11-14 1 24
Assignment 2002-10-01 3 85
Assignment 2003-01-31 3 144
Fees 2004-09-09 1 32
Maintenance Fee Payment 2017-09-12 1 33
Correspondence 2005-09-29 1 30
Fees 2006-09-27 1 36
Prosecution-Amendment 2007-09-07 1 38
Correspondence 2011-08-03 1 42
Maintenance Fee Payment 2018-09-17 1 33
Maintenance Fee Payment 2018-09-17 1 33
Office Letter 2018-09-26 1 28
Refund 2018-10-03 2 72
Prosecution-Amendment 2010-04-20 2 70
Refund 2019-01-02 1 23
Prosecution-Amendment 2010-10-04 21 720
Maintenance Fee Payment 2019-09-23 1 33
Fees 2013-09-25 1 33
Fees 2016-09-14 1 33
Fees 2015-09-21 1 33