U.S. patent application number 10/936937 was filed with the patent office on 2005-02-10 for method for selectively separating blood cells by using lectin.
This patent application is currently assigned to NETECH INC.. Invention is credited to Ihara, Akira, Kitagawa, Michihiro, Saito, Yoshio, Wakamatsu, Daisuke, Yura, Hirofumi.
Application Number | 20050032201 10/936937 |
Document ID | / |
Family ID | 13993424 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050032201 |
Kind Code |
A1 |
Yura, Hirofumi ; et
al. |
February 10, 2005 |
Method for selectively separating blood cells by using lectin
Abstract
The present invention provides a method for selective and
high-yield separation, concentration, and recovery of desired cells
from a blood sample. The method of the present invention is
characterized in that the blood sample is caused to interact with
lectins under conditions in which the cell membranes are inactive
and cell-lectin complexes/non-complexes are formed, the sample
containing these cell-lectin complexes/non-complexes is incubated
together with a substrate, the surface of which is covered with
polymers having carbohydrate chains which are specifically
recognized by the lectins, and the cells are immobilized on the
surface of the substrate via the lectins, and subsequently, the
liquid layer and the solid phase are separated, and the desired
blood cells are recovered from the liquid phase and/or the solid
phase, and these lectins are present in such an amount that
although the cells to be recovered from the solid phase bind to the
solid phase with the polymer, the cells to be recovered from the
liquid phase do not bind to the solid phase with the polymer.
Inventors: |
Yura, Hirofumi;
(kawasaki-shi, JP) ; Saito, Yoshio; (Yokohama-shi,
JP) ; Kitagawa, Michihiro; (Tokyo, JP) ;
Wakamatsu, Daisuke; (Kobe-shi, JP) ; Ihara,
Akira; (Tokyo, JP) |
Correspondence
Address: |
PEARNE & GORDON LLP
1801 EAST 9TH STREET
SUITE 1200
CLEVELAND
OH
44114-3108
US
|
Assignee: |
NETECH INC.
Kawasaki-shi
JP
|
Family ID: |
13993424 |
Appl. No.: |
10/936937 |
Filed: |
September 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10936937 |
Sep 9, 2004 |
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09937510 |
Jan 23, 2002 |
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09937510 |
Jan 23, 2002 |
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PCT/JP00/02011 |
Mar 30, 2000 |
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Current U.S.
Class: |
435/287.1 |
Current CPC
Class: |
G01N 33/56966 20130101;
G01N 33/56972 20130101; C12N 5/0634 20130101; G01N 2333/4724
20130101 |
Class at
Publication: |
435/287.1 |
International
Class: |
C12M 001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 30, 1999 |
JP |
11-090256 |
Claims
What is claimed is:
1. An apparatus for separating and recovering hematopoietic cells
and/or erythroblasts from a blood sample containing differentiated
mature cells, immature hematopoietic cells and erythroblasts
comprising: (1) a device in which said sample interacts with
lectins to form cell-lectin complexes/non-complexes under
conditions in which the cells are rendered inactive, (2) a
substrate on which incubation of said sample containing said
cell-lectin complexes/non-complexes is carried out under said
conditions, said substrate having a surface that is covered with a
synthetic glycoconjugate polymer having carbohydrate moieties that
are specifically recognized by said lectins, said cells being
immobilized on the surface of said substrate via said lectins, and
(3) a device that separates a liquid phase from a solid phase and
in which desired blood cells are recovered from said liquid phase
and/or said solid phase, wherein said lectins are present in an
amount such that they bind to the cells recovered from said solid
phase and immobilize these cells on the surface of the substrate,
but do not immobilize the cells recovered from said liquid phase to
the surface of said substrate.
2. An apparatus according to claim 1 further comprising a device in
which accelerating and stabilizing the immobilization of cells is
carried out by centrifuging said substrate and cells simultaneously
prior to or after the incubation or in which stabilizing the
immobilization of cells is carried out by centrifuging the
substrate on which the cells are immobilized during recovery of the
desired blood cells.
3. An apparatus according to claim 1, wherein said device in which
said sample interacts with said lectins operates under said
conditions under which said cells are rendered inactive comprising:
low temperature conditions of 0.degree. C. or above but less than
37.degree. C., or conditions in which a pharmaceutical agent is
added which suspends cellular respiration.
4. An apparatus according to claim 1, wherein the concentration of
said lectins is within a range of 20 mg/ml or less per cell.
5. An apparatus according to claim 1, wherein said device in which
said sample interacts with said lectins is set to permit said
interaction for a time period within a range of 0 to 120 minutes,
and said incubation on said substrate is carried out for a time
period in a range of 10 to 120 minutes.
6. An apparatus according to claim 1, wherein the substrate is
selected from the group consisting of dishes, flasks, plates,
cuvettes, films, fibers, or beads made of glass, polystyrene,
polycarbonate, polysulfone, polyurethane, or vinyl copolymer, and
that carbohydrate components capable of bonding with said lectins
are treated on the substrate.
7. An apparatus for separating and recovering hematopoietic cells
and/or erythroblasts from a blood sample containing differentiated
mature cells, immature hematopoietic cells and erythroblasts
comprising: (1) means for causing said sample to interact with
lectins to form cell-lectin complexes/non-complexes under
conditions in which the cells are rendered inactive, (2) a
substrate on which incubation of said sample containing said cell
lectin complexes/noncomplexes is carried out under said conditions,
said substrate having a surface that is covered with a synthetic
glycoconjugate polymer having carbohydrate moieties that are
specifically recognized by said lectins, said cells being
immobilized on the surface of said substrate via said lectins, and
(3) means for separating a liquid phase from a solid phase and
recovering desired blood cells from said liquid phase and/or said
solid phase; wherein said lectins are present in an amount such
that they bind to the cells recovered from said solid phase and
immobilize these cells on the surface of the substrate, but do not
immobilize the cells recovered from said liquid phase to the
surface of said substrate.
8. An apparatus according to claim 1 wherein said glycoconjugate
polymer is selected from the group consisting of: Poly(N-p-vinyl
benzyl-[O-.beta.-D-galactopyranosyl-(1.fwdarw.4)-D-gluconamide])
(referred to as PVLA); Poly(N-p-vinyl
benzyl-[O-.alpha.-D-glucopyranosyl-- (1.fwdarw.4)-D-gluconamide])
(referred to as PVMA); Poly(N-p-vinyl
benzyl-[O-.beta.-D-mannopyranosyl-(1.fwdarw.4)-D-mannamide])
(referred to as PVMan); Poly(N-p-vinyl
benzyl-[O-.alpha.-D-galactopyranosyl-(1.alpha..-
fwdarw.6)-D-gluconamide]) (referred to as PVMeA); Poly(N-p-vinyl
benzyl-[O-6-carboxymethyl-.beta.-D-galactopyranosyl-(1.fwdarw.4)--O-D-6-c-
arboxymethyl-gluconamide]) (referred to as PVLACOOH);
Poly(3-O-4'-vinyl benzyl-D-glucose) (referred to as PVG);
Poly(N-p-vinyl
benzyl-[O-2-acetamide-2-deoxy-.beta.-D-glucopyranosyl-(1.fwdarw.4)-O-D-2--
acetamide-2-deoxy-.beta.-D-glucopyranosyl-(1.fwdarw.4)-O-D-2-acetamide-2-d-
eoxy-.beta.-D-gluconamide]); poly(N-p-vinyl
benzyl-[O-D-2-acetamide-2-deox-
y-.beta.-D-glucopyranosyl-(1.fwdarw.4)-O-D-2-acetamide-2-deoxy-.beta.-D-gl-
uconamide]); and mixtures thereof (all referred to as PVGlcNac);
Poly(N-p-vinyl
benzyl-[O-.beta.-D-glucopyranosyl-(1.fwdarw.3)-D-gluconami- de])
(referred to as PVLam); and copolymers and combinations
thereof.
9. An apparatus according to claim 1 wherein said glycoconjugate
polymer is selected from the group consisting of: Poly(N-p-vinyl
benzyl-[O-.beta.-D-galactopyranosyl-(1.fwdarw.4)-D-gluconamide])
having .beta.-galactose residues obtained by polymerizing monomers
synthesized from p-amino methyl styrene and lactose (referred to as
PVLA); Poly(N-p-vinyl
benzyl-[O-.alpha.-D-glucopyranosyl-(1.fwdarw.4)-D-gluconam- ide])
having glucose residues obtained by polymerizing monomers
synthesized from p-amino methyl styrene and maltose (referred to as
PVMA); Poly(N-p-vinyl
benzyl-[O-.beta.-D-mannopyranosyl-(1.fwdarw.4)-D-ma- nnamide])
having mannose residues obtained by polymerizing monomers
synthesized from p-amino methyl styrene and mannobiose (referred to
as PVMan); Poly(N-p-vinyl
benzyl-[O-.alpha.-D-galactopyranosyl-(1.alpha..fwd-
arw.6)-D-gluconamide]) having .alpha.-galactose residues obtained
by the polymerization of monomers synthesized from p-amino methyl
styrene and O-.alpha.-D-galactopyranosyl-(1.fwdarw.6)-D-glucose
(referred to as PVMeA); Poly(N-p-vinyl
benzyl-[0-6-carboxymethyl-.beta.-D-galactopyranosy-
l-(1.fwdarw.4)-O-D-6-carboxymethyl-gluconamide]) having
carboxymethylated-.beta.-galactose residues obtained by the
carboxymethylation of PVLA which is obtained by the polymerization
of monomers synthesized from p-amino methyl styrene and lactose
(referred to as PVLACOOH); Poly(3-O-4'-vinyl benzyl-D-glucose)
having glucose residues obtained by the polymerization of monomers
synthesized from p-chloromethyl styrene and glucose (referred to as
PVG); Poly(N-p-vinyl
benzyl-[O-2-acetamide-2-deoxy-.beta.-D-glucopyranosyl-(1.fwdarw.4)-O-D-2--
acetamide-2-deoxy-.beta.-D-glucopyranosyl-(1.fwdarw.4)-O-D-2-acetamide-2-d-
eoxy-.beta.-D-gluconamide]); poly(N-p-vinyl
benzyl-[O-D-2-acetamide-2-deox-
y-.beta.-D-glucopyranosyl-(1.fwdarw.4)-O-D-2-acetamide-2-deoxy-.beta.-D-gl-
uconamide]); and mixtures thereof having N-acetylglucosamine
residues obtained by the polymerization of monomers synthesized
from p-chloromethyl styrene and N-acetylglucosamine (all referred
to as PVGlcNac); Poly(N-p-vinyl
benzyl-[O-.beta.-D-glucopyranosyl-(1.fwdarw.3)-- D-gluconamide])
having .beta.1.fwdarw.3 glucose residues obtained by polymerizing
monomers synthesized from p-amino methyl styrene and laminaribiose
(referred to as PVLam); and copolymers and combinations
thereof.
10. An apparatus according to claim 9 wherein said glycoconjugate
polymer is a copolymer formed by polymerizing monomers one of which
comprises an azide group.
11. An apparatus according to claim 10 wherein said copolymer is
selected from the group consisting of: poly(3-azide
styrene-co-{N-p-vinyl
benzyl-[O-.beta.-D-galactopyranosyl-(1.fwdarw.4)-D-gluconamide]})
(which is referred to as AZ-PVLA); poly(3-azide
styrene-co-{N-p-vinyl
benzyl-[O-.alpha.-D-glucopyranosyl-(1.fwdarw.4)-D-gluconamide]})
(which is referred to as AZ-PVMA); and combinations thereof.
12. A device for separating and recovering hematopoietic cells
and/or erythroblasts from a blood sample containing differentiated
mature cells, immature hematopoietic cells and erythroblasts,
comprising cell-lectin complexes formed by interaction of lectins
and said sample in which the cells are rendered inactive, and a
substrate having a surface that is covered with a synthetic
glycoconjugate polymer having carbohydrate moieties, said
cell-lectin complexes being immobilized to said carbohydrate
moieties via said lectins, wherein said lectins are present in an
amount such that they bind to cells recovered from a solid phase
that has been separated from a liquid phase, and immobilize these
cells on the surface of the substrate, but do not immobilize the
cells recovered from said liquid phase to the surface of said
substrate.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for the separation
of blood cells using lectins, and in particular, relates to a
method for the selective separation and recovery of desired blood
cells, via lectin, from a sample containing both mature cells and
immature cells contained in peripheral blood, bone marrow fluid,
umbilical cord blood, or the like.
BACKGROUND OF THE INVENTION
[0002] Hematopoietic stem cells are cells which combine the
potential of multi-differentiation and autoreproduction. The
potential of autoreproduction is most important for hematopoiesis
in order that blood cells not become exhausted over the course of a
lifetime. With respect to the ability of the hematopoietic stem
cells to multi-differentiate, as shown in FIG. 1, stem cells
differentiate into myeloid stem cells and lymphatic stem cells, and
these further differentiate into platelets, (mature) erythrocytes,
granulocytes, monocytes, or the like from the myeloid stem cells,
while blood cells such as T cells or B cells or the like are
produced from the lymphatic stem cells.
[0003] Blood cells have a lifetime and are consumed in accordance
with a variety of physiological needs, so that it is necessary that
the blood cells be appropriately replenished by differentiation
from stem cells. In patients suffering from, for example, acute
myelogenous leukemia, there are irregularities in the
differentiated functional blood cells themselves, as well as in the
stem cell differentiation, so that the replenishment of functional
red blood cells, white blood cells, platelets and the like is
difficult. The transplantation of hematopoietic stem cells offers a
treatment method for such blood diseases which does not have the
side effects of chemotherapy and results in the recovery of
functional hematopoiesis through the differentiation and
regeneration of these cells. However, despite these advantages, the
acquisition of stem cells is difficult, as they are almost all
distributed in the bone marrow. Although placental blood and
umbilical cord blood are comparatively enriched in stem cells, so
that a less invasive method of obtaining them is possible, they can
be used only in childbirth. On the other hand, in the peripheral
blood which can be obtained from a donor in the least invasive way,
the amounts of stem cells are further reduced which makes the
peripheral blood less practical.
[0004] Furthermore, in transplanted blood cells containing stem
cells, immunological rejection reaction, termed graft-versus-host
disease (GVHD), may be induced when the HLA type of the patient and
donor do not match. Accordingly, in order to conduct effective and
safe transplantation of stem cells, it is necessary to obtain a
stem cell sample from which lymphocyte fractions which give rise to
GVHD are removed.
[0005] Furthermore, if pure stem cells could be isolated, they
could be effectively stimulated and expanded using cytokines.
Consequently, stem cell isolation could contribute to the
development of stem cell banks in which such cells were stored for
later use.
[0006] On the other hand, in concert with the recent development in
genetic manipulation techniques, efforts have been made to conduct
prenatal gene diagnosis for fetal nucleated cells. Fetal nucleated
cells for diagnostic use which are currently clinically employed,
are collected through invasive methods such as amniocentesis,
chorionic villous sampling, and fetal blood collection, and these
carry the risk of infection and amniorrhexis. It is conventionally
known that fetal cells are admixed in the maternal blood, and the
use of maternal peripheral blood to obtain fetal nucleated cells as
a non-invasive collection has been considered; however, nucleated
red blood cells (NRBC) being likely fetal cells are contained in
the maternal peripheral blood in very small amounts, being only 1
in 10.sup.5-10.sup.7 of the total nucleated cells in the peripheral
blood, so that the key to genetic diagnosis of fetal cells has been
how to concentrate, separate, or identify such cells.
[0007] In addition, it is known that gene diagnosis is effective
for the therapy of leukemia. For example, since leukemia includes
various types such as myeloid type in which hematopoietic cells
themselves are pathologic or other type in which peripheral
lymphocytes or monocytes become malignant, it is necessary to
identify the type of leukemia for determining optimal dosing or
therapeutic regimen. Moreover, genetic examination is needed to
know the stage of differentiation of blood cells in which a
carcinogenic factor is induced because the detection of the stage
in which carcinogenic cells occur would contribute not only to the
treatment of leukemia but also to clinically important matters such
as prevention or recurrence of cancer. In such an examination of
leukemia, it is also necessary to simplify and make effective the
gene diagnosis in each differentiation stage by selecting and
purifying immature hematopoietic blood cells and proliferating and
differentiating them with cytokines.
[0008] The present inventors have conducted research which focused
on the specific interactions between carbohydrates and other
biological substances, and have filed a patent application on a
method for selectively binding lectins, carbohydrate-specific
proteins, to a solid support, such as a dish or the like, covered
with synthetic glycoconjugate polymers including carbohydrate
moieties (Japanese Patent Application No. Hei 8-59695).
[0009] On the other hand, as is shown in FIG. 1, hematocytes
derived from hematopoietic stem cells express a variety of
carbohydrate chains on the cell surface in accordance with the
maturation thereof. In FIG. 1, the designation "Gal" indicates
galactose, "Glu" indicates glucose, and "Lac" indicates lactose
(Glu-Gal). In the patent application referred to above, it is
disclosed that mature human erythrocytes expressing galactose are
preferentially attached to the surface of the substrate covered
with the glycoconjugate polymer including galactose, via a lectin
(Allo-A) which recognizes galactose.
[0010] The present inventors have now thoroughly explored a control
method for blood cell immobilization on a solid support covered
with glycoconjugate polymers via lectins, and have discovered that
by means of the incubation temperature or the amount of lectins
added, a specific system of interactions among the cells and/or the
carbohydrate moieties in the polymers and the lectins can be
produced; the present invention was arrived at on this basis.
SUMMARY OF THE INVENTION
[0011] It is an object of the present invention to provide a method
by which desired immature blood cells or differentiated mature
cells may be selectively, and with high yield, separated,
concentrated, and recovered, using lectins, and to provide a
separation apparatus employing this method.
[0012] This object can be accomplished by a method for selectively
separating and recovering hamatopoietic cells and/or erythroblasts
from a blood sample containing differentiated mature cells,
immature hematopoietic cells and erythroblasts, characterized in
that the method comprises the following steps:
[0013] (1) a step for causing said sample to interact with lectins
to form cell-lectin complexes/non-complexes under conditions in
which the cells are rendered inactive,
[0014] (2) a step for incubating a sample containing said
cell-lectin complexes/non-complexes under said conditions with a
substrate, the surface of which is covered with a synthetic
glycoconjugate polymer having carbohydrate moieties specifically
recognized by said lectins, and immobilizing said cells on the
surface of said substrate via lectins, and
[0015] (3) a step for separating the liquid phase from the solid
phase, and recovering desired blood cells from said liquid phase
and/or said solid phase; and optionally
[0016] (4) a step for accelerating and stabilizing the
immobilization of cells by centrifuging said substrate and cells
simultaneously prior to or after the incubation or a step for
stabilizing the immobilization of cells by centrifuging the
substrate on which the cells are immobilized during the recovering
step,
[0017] and in that said lectins are present in an amount such that
they bind to the cells recovered from said solid phase and
immobilize these cells on the surface of the substrate, but do not
immobilize the cells recovered from said liquid phase to the
surface of said substrate.
[0018] In the method for selectively separating, the conditions
under which the cells are rendered inactive may be low temperature
conditions of 0.degree. C. or above but less than 37.degree. C., or
conditions in which a pharmaceutical agent is added which suspends
cellular respiration. Furthermore, by adjusting the lectin
concentration or incubation time, it becomes possible to obtain
high-level selective separation which has not been accomplished
until now.
[0019] Furthermore, the present invention also provides an
apparatus for use in the above-described selectively separating
method.
[0020] The separating method and apparatus of the present invention
are based on cell-lectin and glycoconjugate-lectin interactions,
and make it possible to selectively separate cells, in particular
specific cells having clinically important significance such as
hematopoietic cells or NRBC.
BRIEF DESCRIPTION OF DRAWINGS
[0021] FIG. 1 is a systematic diagram showing the state of
hematopoietic stem cell differentiation.
[0022] FIG. 2 is a graph showing the results of the selective
concentration of erythroblasts by means of changes in the lectin
concentration in Example 6.
DETAILED DESCRIPTIONS FOR PREFERRED EMBODIMENTS
[0023] Hereinbelow, the present invention will be discussed in
detail.
[0024] In the separation method of the present invention, as a
first stage, a sample containing cells which are to be immobilized
in a solid phase is caused to interact with lectins which recognize
carbohydrates expressed on these cells, and cell-lectin
complexes/non-complexes are formed. Here, what is meant by
cell-lectin complexes/non-complexes is a coexistent state in which
both complexes in which cells and lectins are bound to one another,
and free and unbound cells and lectins (non-complexes) are present.
This step is conducted under conditions such that the cells are
rendered inactive.
[0025] Here, what is meant by conditions in which the cells are
inactive are conditions in which the mobility of the cell membranes
and the self-adhesiveness thereof are lowered, and such conditions
are typically achieved by adjusting the temperatures to within a
range of from 0.degree. C. to less than 37.degree. C., preferably
within a range of 0-36.degree. C., more preferably within a range
of 4-30.degree. C., and most preferably within a range of
4-22.degree. C. However, these conditions are not necessarily
limited to the low temperature adjustment described above; such
conditions may also be achieved by, for example, adding a
pharmaceutical agent which suspends cellular respiration at
37.degree. C., such as sodium azide or the like.
[0026] The lectins are employed to recognize carbohydrates
expressed on cells, which are to be immobilized. For example, as is
shown in FIG. 1, when mature leukocytes, platelets, or erythrocytes
of the peripheral blood which express galactose or glucose are to
be immobilized, then lectins which recognize galactose, such as
SBA, PNA, ECL, Allo A, VAA, or the like, or lectins which recognize
glucose such as Con A, LcH, PSA, or the like, are selected. When
cells expressing mannose are to be immobilized, then lectins which
recognize mannose, such as LCA, GNA, CPA, or the like, are
selected.
[0027] The amount of such lectins added varies based on the type of
cells which are to be immobilized; fundamentally, the amount should
be such that, in the following second stage incubation, the cells
which are to be immobilized (the cells which are later to be
recovered from the solid phase) are bonded to the surface of the
substrate covered with the polymers, while the cells which are not
to be immobilized (the cells which are later to be recovered from
the liquid phase) are not bonded to the surface of the substrate.
By means of specifying the amount of lectin added, it is possible
to control, for example, the selectivity with respect to the
maturity of the cells such as leukocytes or the like, or the
selectivity among cell types such as leukocytes, erythrocytes, and
the like. Concretely, the concentration is adjusted to 20 mg/ml or
less with respect to one cell or more. This amount added varies
based on the type of lectins as well, so that for example, when SBA
is employed as the lectin, an amount of 5 mg/ml is sufficient.
[0028] In particular, when a polymer including a lactose structure,
which has a .beta.-bond galactose terminal, or a mellibiose
structure, which has an .alpha.-bond galactose terminal, is
employed as the glycoconjugate polymer, and when CD34-negative
mature cells are to be predominantly immobilized with controlling
the immobilization of immature CD34-positive cells, the
concentration of lectin added with respect to a sample containing
2.times.10.sup.6 cells may be within a range of 0.001-0.9 mg/ml,
preferably within a range of 0.002-0.1 mg/ml, and more preferably
within a range of 0.025-0.05 mg/ml. Furthermore, when red blood
cell (NRBC) are to be selectively immobilized, and other cells such
as leukocytes or the like are not to be immobilized, then the
concentration of lectin with respect to a sample containing
2.times.10.sup.6 cells may be within a range of 0.001-0.3 mg/ml,
preferably within a range of 0.002-0.05 mg/ml, and more preferably
within a range of 0.004-0.025 mg/ml.
[0029] The incubation period in the first stage is not particularly
restricted; it should be set so that the cells and the lectins
interact prior to the following second stage, and form cell-lectin
complexes/non-complexes, and typically this will be within a range
of 0-120 minutes, preferably within a range of 0-90 minutes, and
more preferably within a range of 0-60 minutes. Here, what is meant
by "0 minutes" is a transfer to the second stage immediately after
conducting the first stage. As a result, the cells which are to be
immobilized and the lectins form cell-lectin
complexes/non-complexes.
[0030] Next, as a second stage in the separation method of the
present invention, the sample containing the cell-lectin
complexes/non-complexes described above is incubated, under
conditions in which the cells are inactive, on a substrate, the
surface of which is covered with the glycoconjugate polymers having
carbohydrates which are specifically recognized by the lectins.
[0031] The substrate employed here may be selected from substrates
conventionally employed for cell cultivation, such as dishes,
flasks, plates, cuvettes, films, fibers, beads, separable chamber
slides, or the like; it is possible to use substrates having a
variety of shapes depending on the use.
[0032] This substrate may be made of inorganic materials such as
glass, silica, or the like, or organic materials such as
polystyrene, polycarbonate, polysulfone, polyurethane, vinyl
copolymers, or the like, as well as composite materials formed
therefrom; however, a material which is resistant to the degree of
heat required for sterilization and which is water-resistant is
preferably used. In particular, synthetic polymeric materials are
preferable from the point of view of cost and moldability, and
hydrophilic materials are preferable from the point of view of the
covering effectively of the glycoconjugate polymers. When, for
example, a glycoconjugate polymer containing a main chain made of
polystyrene or its derivatives is employed, the use of a substrate
material having polystyrene or its derivatives is preferable for
the cover.
[0033] The surface of the substrate described above is covered with
the glycoconjugate polymers having carbohydrate moieties, which are
specifically recognized by the lectins used in the first stage.
[0034] Examples of such polymers include the following:
[0035] Poly(N-p-vinyl
benzyl-[O-.beta.-D-galactopyranosyl-(1.fwdarw.4)-D-g- luconamide])
having .beta.-galactose residues obtained by polymerizing monomers
synthesized from p-amino methyl styrene and lactose (referred to as
PVLA);
[0036] Poly(N-p-vinyl
benzyl-[O-.alpha.-D-glucopyranosyl-(1.fwdarw.4)-D-gl- uconamide])
having glucose residues obtained by polymerizing monomers
synthesized from p-amino methylstyrene and maltose (referred to as
PVMA);
[0037] Poly(N-p-vinyl
benzyl-[O-.beta.-D-mannopyranosyl-(1.fwdarw.4)-D-man- namide])
having mannose residues obtained by polymerizing monomers
synthesized from p-amino methyl styrene and mannobiose (referred to
as PVMan);
[0038] Poly(N-p-vinyl
benzyl-[O-.alpha.-D-galactopyranosyl-(1.alpha..fwdar-
w.6)-D-gluconamide]) having .alpha.-galactose residues obtained by
the polymerization of monomers synthesized from p-amino methyl
styrene and O-.alpha.-D-galactopyranosyl-(1.fwdarw.6)-D-glucose
(referred to as PVMeA);
[0039] Poly(N-p-vinyl
benzyl-[O-6-carboxymethyl-.beta.-D-galactopyranosyl--
(1.fwdarw.4)-O-D-6-carboxymethyl-gluconamide]) having
carboxymethylated-.beta.-galactose residues obtained by the
carboxymethylation of PVLA which is obtained by the polymerization
of monomers synthesized from p-amino methyl styrene and lactose
(referred to as PVLACOOH);
[0040] Poly(3-O-4'-vinyl benzyl-D-glucose) having glucose residues
obtained by the polymerization of monomers synthesized from
p-chloromethyl styrene and glucose (referred to as PVG);
[0041] Poly(N-p-vinyl
benzyl-[O-2-acetamide-2-deoxy-.beta.-D-glucopyranosy-
l-(1.fwdarw.4)-O-D-2-acetamide-2-deoxy-.beta.-D-glucopyranosyl-(1.fwdarw.4-
)-O-D-2-acetamide-2-deoxy-.beta.-D-gluconamide]), poly(N-p-vinyl
benzyl-[O-D-2-acetamide-2-deoxy-.beta.-D-glucopyranosyl-(1.fwdarw.4)-O-D--
2-acetamide-2-deoxy-.beta.-D-gluconamide]) and mixtures thereof
having N-acetylglucosamine residues obtained by the polymerization
of monomers synthesized from p-chloromethyl styrene and
N-acetylglucosamine (all termed PVGlcNac); and
[0042] Poly(N-p-vinyl
benzyl-[O-.beta.-D-glucopyranosyl-(1.fwdarw.3)-D-glu- conamide])
having .beta.1.fwdarw.3 glucose residues obtained by polymerizing
monomers synthesized from p-amino methyl styrene and laminaribiose
(termed PVLam).
[0043] These glycoconjugate polymers may be homopolymers as
described above or may be copolymers with other monomers. For
example, copolymers with monomers having azide groups, which are
photoreactive functional groups, are preferable in that they
facilitate the formation of a covalent bond with the substrate
surface by means of the application of light. Examples of
glycoconjugate polymers into which azide groups are introduced
include: poly(3-azide styrene-co-{N-p-vinyl
benzyl-[O-.beta.-D-galactopyranosyl-(1.fwdarw.4)-D-gluconamide]}),
which is a copolymer with the PVLA described above (termed
AZ-PVLA); and poly(3-azide styrene-co-{N-p-vinyl
benzyl-[O-.beta.-D-glucopyranosyl-(1.f- wdarw.4)-D-gluconamide]}),
which is a copolymer with the PVMA described above (referred to as
AZ-PVMA), and the like.
[0044] When the incubation in the second stage is conducted in a
dish or flask used for cell culturing under the conditions same as
described for the first stage above, then the incubation period is
typically within a range of 10-120 minutes, preferably within a
range of 20-90 minutes, and more preferably within a range of 30-70
minutes. As a result, the cell-lectin complexes to be immobilized
are attached to the substrate via the glycoconjugate polymers on
the surface of the substrate.
[0045] On the other hand, under conditions in which the cell
precipitation period can be ignored in a column filled with beads
or non-woven fabric, this period may be further shortened. The
shortening of the period can also be attained by acceleration and
stabilization of the precipitation and immobilization of cells by
means of centrifuging the substrate and cells simultaneously prior
to or after the incubation of the second stage. The centrifuging
period is sufficient within a range of 30 minutes or less, and it
preferably be 10 minutes or less from the point of view of
shortening time. The centrifuging force will vary depending on the
type of cells to be immobilized or types or concentrations of
lectins used, and in general it preferably be in a range of 30-450
G.
[0046] Next, as a third stage of the separation method of the
present invention, the solid phase which is immobilized on the
substrate, and the liquid phase which remains unimmobilized are
separated. In the case in which a column filled with beads or
plates covered with the carbohydrates is employed, a sample
containing the cell-lectin complexes/non-complexes obtained in the
first stage may simply be introduced into the column, and thereby,
it is possible to separate the solid phase by recovering the liquid
phase from the outlet of the column.
[0047] When chromosomes of the cells attached on a substrate, such
as a chamber slide or dish, are examined, the liquid phase staying
unattached in the above recovering step is removed, and the
substrate on which cells are attached is then centrifuged to
stabilize the immobilization of the cells which results in a
uniform immobilized image. The centrifuging period is sufficient
within a range of 30 minutes or less, and it preferably be 10
minutes or less from the point of view of shortening time. The
centrifuging force will vary depending on the type of cells to be
immobilized or types or concentrations of lectins used, and in
general it is preferably 30 G or more, more preferably in a range
of 100-400 G, and further preferably in a range up to 1,000 G.
[0048] More specifically, in the case in which, for example, PVLA
is employed as the glycoconjugate polymer, and SBA or the like is
employed as the galactose-specific lectin in order to recover
desired hematopoietic cells from liquid and/or solid phase, then
the ease of attachment goes in the order of mature erythrocytes,
NRBC>leukocytes>immature CD34-positive cells. Accordingly, if
the amount of lectin is reduced, the CD34-positive cells will first
become unattached, and then the leukocytes will become unattached,
and finally, the mature erythrocytes and NRBC will be the only
cells which are selectively attached. In other words, if the
separation method of the present invention is conducted using an
amount of added lectin such that only CD34-positive cells remain
unattached, then the CD34-positive cells will be selectively
contained in the liquid phase, and it will be possible to recover
these CD34-positive cells with a high degree of purity.
Furthermore, if the amount of added lectin is further reduced, and
only mature erythrocytes and NRBCs are attached, then it is
possible to selectively recover mature erythrocytes and NRBCs from
the solid phase.
[0049] In this separation method of the present invention, it is
also possible to remove granulocytes and leukocytes and the like
from the blood sample obtained in advance prior to the first stage,
so as to concentrate the desired cells. In such a case,
multi-purpose high-density liquid set to specific gravity of about
1.077 such as Ficoll Paque, Histo Paque, Percall, or the like may
be generally used, while in the present invention, those having a
specific gravity of 1.085-1.10 are particularly used in a
pretreatment for separating and concentrating NRBC from a blood
sample. In practice, the present inventors confirmed that when a
high-density liquid, such as Ficoll Paque, Histo Paque, Percall, or
the like, having a specific gravity of 1.095 is used, the resulting
recovered amounts of NRBC via lectin were improved about 1.5 times
larger than those obtained using a conventional high-density liquid
having a specific gravity of 1.077. Although many investigators
have considered the effects of these pretreatments for a long time,
no conclusion has been obtained due to variations among individuals
and the like. Therefore, the other conditions may also be
appropriately employed in the selective separating method of the
present invention.
[0050] Furthermore, as shown in FIG. 1, it is also of course
possible to conduct the method in the same way using synthetic
glycoconjugate polymer of glucose family and lectin.
[0051] In general, negative separation, in which cells other than
those which are desired are immobilized, and positive separation,
in which the cells which are desired are immobilized and
concentrated, are known as methods for separating cells; however,
the separation method of the present invention makes use of both
negative and positive separation by appropriately adjusting the
concentration of lectins.
[0052] In the selective separation of hematopoietic stem cells
which are present in very small amounts, in order to reduce the
number of the cells which are wasted, the method described above
may be repeated a number of times to make it possible to increase
the yield of the desired cells.
[0053] The blood sample which is separated and refined by means of
the separation method of the present invention may be from any
source, including peripheral blood; however, in the selective
recovery of stem cells, bone marrow fluid, umbilical blood, or
placental blood is preferable. Furthermore, in the selective
recovery of NRBCs, umbilical blood or maternal blood is
preferable.
EXAMPLES
[0054] Hereinbelow, a case will be concretely discussed in which,
following the separation method of the present invention, and
employing Az-PVLA as a glycoconjugate polymer, and using a
phosphate-buffered physiological saline (PBS) supplemented with
0.1% by weight of bovine serum albumin as a cell suspension. In
this case, immature hematopoietic stem cells are selectively
separated and recovered.
[0055] 1: Incubation Conditions
Example 1
Temperature Effects (1)
[0056] First stage: Cord blood mononucleated cells monocytes were
obtained as cells treated with ammonium chloride after
centrifugation on Ficoll Paque. In a tube made by polypropylene,
PBS containing a variety of concentrations of SBA (lectin specific
for galactose) was added to a suspension of the mononucleated cells
derived from cord blood of 2.times.10.sup.6 cells per ml, and the
mixture was incubated at a temperature of 4.degree. C. for a period
of 30 minutes and was gently stirred at intervals of 5 minutes.
[0057] Second stage: after the completion of the incubation
described above, the suspension was transferred to a dish having a
diameter of 35 mm which was coated with AZ-PVLA, the tube was
further rinsed with 1 ml of the isotonic salt solution described
above, and this rinse liquid was also added to the dish, and
incubation was conducted at a variety of temperatures from
4.degree. C. to 37.degree. C. for a period of 60 minutes.
Alternatively, the dish and cells were centrifuged for a
predetermined period at 90 G in place of the incubation for 60
minutes, or the substrate and cells were incubated for 15 minutes
and then centrifuged for a predetermined period in place of the
incubation for 60 minutes. In addition, both of the centrifuging
treatments were conducted concomitantly.
[0058] Third stage: after stirring, the suspension liquid was
recovered, washing was conducted with 1 ml of the PBS, and the
solid phase (dish) and liquid phase (suspended liquid) were
separated.
[0059] The recovered cell count in the cellular suspension liquid
obtained was measured using an automated blood cell counter, and
the proportion (attachment ratio) of attaching cells with respect
to the number of cells used was calculated. As a result, it was
discovered that there was a trend for the attachment ratio to
increase as the amount of lectin added increased at all
temperatures. The amount of added lectin (SBA), which was minimally
necessary in order to cause the adhesion of 80% of the
mononucleated cells at each temperature, was as shown in Table 1
below.
1TABLE 1 Processing Minimum Amount of Added SBA Required to Cause
80% Temperature of Mononucleated cell Attachment to applying 2
.times. 10.sup.6 cells 37.degree. C. 1.0 mg 30.degree. C. 0.5 mg
10.degree. C. 0.05 mg 4.degree. C. 0.025 mg
[0060] From these results, it can be seen that the amount of added
lectin (SBA) necessary to cause the attachment of 80% of the
mononucleated cells decreased along with a decrease in temperature,
so that in other words, by reducing the incubation temperature, the
attachment efficiency could be increased, so that attachment and
separation became possible with small amounts of lectin. Under the
processing temperature at 4.degree. C., a cellular attachment of
approximately 50% was observed at 0.01 mg. The fact that this
cellular attachment was specific for carbohydrate via lectin was
confirmed by the fact that, by adding the galactose solution in
various concentrations to the dish, the cellular adhesion was
inhibited by 60-90% both at 4.degree. C. and 37.degree. C.
[0061] In addition, in the case in which centrifuging at 90 G was
conducted in place of the incubation for 60 minutes, centrifuging
for not less than 3 minutes provided the same cell-attachment as
that obtained by the incubation for 60 minutes. Furthermore, in the
case in which incubation for 15 minutes followed by centrifuging at
90 G was conducted in place of the incubation for 60 minutes,
stable cell-attachment was obtained by centrifuging for not less
than 2 minutes. These centrifuging treatments were able to
facilitate and stabilize the selective attachment of the cells via
lectins and contributed to shortening processing period, unless
such centrifuging was so excessive to destroy the cells resulting
in unselective adhesion.
Example 2
Temperature Effects (2)
[0062] A procedure was followed under conditions which were
identical to those of Example 1, with the exception that the
lectins employed were PNA and ECL (both of which are
galactose-specific), and the results shown in Table 2 below were
obtained.
2 TABLE 2 Cellular Attachment Ratio Amount of Processing Processing
Lectin Added Temperature Temperature Lectin (mg/2 .times. 10.sup.6
cells) 4.degree. C. 37.degree. C. PNA 0.72 73 44 ECL 0.02 81 58
[0063] From the results in Table 2, it became clear that,
irrespective of the type of lectin employed, a relationship was
present between the cellular attachment ratio and the incubation
temperature, as shown above.
Example 3
Contents of Temperature Effects
[0064] The same type of experiment was conducted under conditions
identical to those of the incubation at 37.degree. C. in Example 1,
and sodium azide was added to the cell suspension. The results are
shown in Table 3. Here, the attachment of the cells is expressed in
terms of the proportion of cells recovered which were not attached
(the recovery ratio).
3TABLE 3 Incubation Conditions (Amount of SBA Added: 1 mg/2 .times.
10.sup.6 cells) Recovery Ratio (%) Incubation temperature of
37.degree. C. 14.8 Incubation temperature of 37.degree. C. + 3.9 10
mM sodium azide Incubation Temperature of 4.degree. C. 6.7
[0065] From the results shown above, it can be seen that the
cellular attachment via lectin increases with a decrease in
temperature; however, this phenomenon is also observed if sodium
azide, which is known to suppress metabolic activities, is added
even if the temperature is not reduced. That is to say, the
temperature-dependent affinity of the lectin for the cells is
affected by the cells affected by the cellular membrane mobility,
and a tendency is observed for the affinity to increase as the
membrane mobility decreases.
[0066] 2. Selective Affinity
Example 4
Selective Affinity Based on the Maturity of Leukocytes
[0067] Immature leukocytes express a surface marker termed CD34 on
the surface thereof (they are CD34-positive) and are known to
become CD34-negative as they mature. Conventionally, the selective
immobilizing of immature cells was conducted using CD34 antibodies.
Here, on solid surfaces with PVLA having a .beta.-bond galactose
terminus attached (at incubation temperatures of 4.degree. C. and
37.degree. C.), and PVMeA having an .alpha.-bond galactose terminus
attached (at an incubation temperature of 4.degree. C.), selective
attachment was investigated using various amounts of added lectin.
The results thereof are shown in Table 4. The described values of
SBA in the table represent the amount added with respect to
2.times.10.sup.6 cells.
4 TABLE 4 Cellular Attachment Ratio (%) Leukocyte SBA 0.025 mg SBA
0.05 mg SBA 1 mg Maturity PVMeA (4.degree. C.) PVLA (4.degree. C.)
PVLA (37.degree. C.) Immature, CD34- 20 15 12 positive cells
Mature, CD34- 76 73 70 negative cells
[0068] From the results above, it was discovered that by means of
the separation method of the present invention using lectin, it is
possible to selectively separate CD34-positive immature cells and
CD34-negative mature cells. Moreover, by means of setting the
incubation temperature to a low temperature, the amount of added
lectin required was reduced to approximately {fraction (1/20)} of
that formerly required. Furthermore, when PVMeA was employed as the
glycoconjugate polymer, in comparison with the case in which PVLA
was employed, selective attachment was obtained even when the
amount of added lectin was further reduced.
[0069] It is generally known that SBA has a stronger affinity to
galactose of .alpha.-bond type. Therefore, the results, in which
the selective cell attachment to PVMeA having .alpha.-bond type
galactose terminal was observed at lower lectin concentration,
demonstrate that the separating method of the present inventors is
based on quite specific affinity of lectins. Furthermore, the
present method clarified that the low-temperature-incubation is an
effective process which can strongly enforce the affinity of
lectins.
Example 5
Selective Affinity among Blood Cells
[0070] An experiment was conducted which was identical to that of
Example 1 and the incubation temperature was set at 4.degree. C.,
and the amount of added lectin required to cause the attachment of
95% of erythrocytes and of leukocytes derived from umbilical cord
blood was determined. In this case, the hemolysis was not
conducted. The results thereof are shown in Table 5 below.
5TABLE 5 Amount of Added Lectin Required to Cause 95% or more Cells
of Erythrocytes or Leukocytes to Attach (2 .times. 10.sup.6 cells)
Leukocytes 300 micrograms or more Erythrocytes 50 micrograms or
more
[0071] As shown in Table 5, in comparing the attachment via lectin
of the erythrocytes and leukocytes, the erythrocytes showed higher
affinity, and were capable of attachment at lower levels of added
lectin.
Example 6
Separation of Umbilical Cord Blood Erythroblasts
[0072] The incubation temperature was set to 4.degree. C., and the
amount of lectin (SBA) added was altered to a very small amount of
50 micrograms or less per 2.times.10.sup.6 cells, and the cells
attaching to a dish covered with PVLA were investigated as in
Example 5. In addition, the results obtained by centrifuging the
dish and attached cells at 450 G after separating the solid phase
from liquid phase in the third step were compared.
[0073] The method employed in this investigation was such that the
attaching cells were allowed to dry on the dish, cells were then
stained with hematoxylin and erythrosin, and positively stained
erythroblasts were counted, and 100 cells in a randomly selected
area were counted and the number of erythroblasts contained therein
was evaluated. The results thereof are shown in FIG. 2. In the
figure, PRE indicates a comparative example in which a large amount
(300 micrograms) of lectin was added and almost all cells were
caused to attach.
[0074] If the amount of lectin added is decreased, and the
attachment of leukocytes which have a lower affinity for the lectin
is preferentially reduced, then mature erythrocytes and
erythroblasts are selectively caused to attach to the dish.
Accordingly, it is possible to detect erythroblasts which are
present at low levels in umbilical cord blood, at a high
probability of 1 or more/100 cells. Furthermore, when centrifuging
at 450 G for not less than 3 minutes was conducted after separating
the solid phase from liquid phase, the attached cells exhibited
uniform spherical shapes which provided good staining sensitivity
and therefore made it easy to visually recognize erythroblasts with
a microscope. However, when the centrifuging treatment was not
sufficient (for example, in a case wherein centrifuging force was
too small or centrifuging period was too short), uniform cell
staining image could not be obtained which resulted in difficulties
in visual recognition of desired cells with a microscope.
Accordingly, it was found that it is very important to centrifuging
the cells attached to the substrate with appropriate conditions
when one intends to improve the efficacy of a cytological
examination, which requires detection of fine nucleus structure by
cell staining.
Example 7
Concentration of Fetal Erythroblasts in the Maternal Blood
[0075] Following the method of Example 6, a cell fraction separated
by the Ficoll Paque was recovered from maternal blood, 10
micrograms of a lectin (SBA) was added with respect to
2.times.10.sup.6 cells, and this was incubated for a period of 30
minutes at a temperature of 18.degree. C. in a PVMeA-covered
separable slide chamber. For the purposes of comparison, a case was
also evaluated in which the amount of lectin added was 300
micrograms. The experimental results for 20 examples are shown in
Table 6 below.
6TABLE 6 Number of Positively Stained Erythroblasts Amount of
Lectin Added Adhered to the Dish 10 micrograms 300 micrograms 0-1 1
instance 19 instances* 2-5 3 instances 1 instance* 6-10 7 instances
-- 11-30 8 instances -- 31 or more 1 instance -- *When 300
micrograms was added, too many nucleated leukocytes other than
erythroblasts were caused to attach, so that a miss count
occurred.
[0076] In consideration with the above results, the optimal amounts
of lectin (SBA) to be added for detecting fetal erythroblasts were
estimated. The results estimated from 20 instances are indicated in
the following Table 7. In each case, a maternal body was examined
based on informed consent by echo imaging to ascertain that she
carries healthy boy. The erythroblasts separated from the collected
maternal blood were examined by FISH assay using Aneu Vysion Assay
Kit (VYSIS, INC.) to detect a Y-probe.
7 TABLE 7 The Amount of Lectin Added (.mu.g) 2 4 8 12 16 The Number
of NRBC detected 0 0.16 1.00 1.07 0.97 (relative value when the
number of NRBC detected with 8 .mu.g of lectin is assumed as
1.00)
[0077] These results show that when the amount of lectin added is
decreased and the attachment of leukocytes is reduced, it is
possible to selectively accumulate erythroblasts, and it is
possible to efficiently detect erythroblasts, which are useful in
genetic diagnosis, from maternal blood. In addition, it was found
that there was an apparent lower-limit of the amount of lectin to
be added, and that the loss of erythroblasts was reduced when 8
.mu.g or more of 1 ectin was added. In such cases, contamination
with nucleated cells or leukocyte was gradually increased when the
amount of lectin exceeded 20 .mu.g, the contamination occurred so
abundantly that the recognition of NRBC became difficult when the
amount of lectin reached to 32 .mu.g or more. Furthermore, these
results were reproducible when PVLA was employed as the
glycoconjugate polymer.
[0078] On the other hand, with the maternal blood samples
containing small amounts of erythrocyte components such as
erythroblasts, good erythrocyte selective attachment was reproduced
without the incubation with lectin at the first stage.
[0079] Furthermore, 8 samples of maternal bodies who carries a boy
were examined at this time, and Y-probes specific for boy were
detected in the 8 samples which means that fetal cells can be
recovered from maternal blood with high yield. Therefore, it was
found that the separation method for nucleated erythroblasts using
a lectin of the invention is an effective means for detecting fetal
cells from maternal blood with no- or low-invasion and examining
the fatal chromosomes.
[0080] The fractions of CD34-positive cells concentrated in
accordance with Example 4 were recovered, and their colony-forming
abilities were compared using commercially available assay kit
(MethoCult GF H4434, Stem Cell Technologies Inc.). As a result, the
CD34-positive cells concentrated by the separation method via
lectin of the invention exhibited colony-forming abilities of 8.8
times larger than that obtained without such separation. These
results demonstrate that the treatment with lectin can effectively
increase the hematopoietic cells without affecting their subsequent
colony-forming abilities. Accordingly, it is believed that the cell
separating method of the invention can provide a transplant graft
with reduced lymphocyte, which can alleviate GVHD, to a patient in
need of stem cell transplant, and therefore, the separating method
of the invention can be used in effective detection of oncogenes
derived in each stage of differentiation of leukemia cells.
INDUSTRIAL APPLICABILITY
[0081] As described in detail above, the separation method and
separation apparatus of the present invention are based on
interaction among cells-lectins and glycoconjugate
polymers-lectins. In greater detail, the present invention utilizes
the fact that the changes in attachment properties depending on the
state of activity of cells or the amount of lectin added, and
changes in attachment selectivity depending of the type of cells
were observed in the above interactions. By employing the
separation method and separation apparatus of the present
invention, it is possible to selectively separate and recover cells
which have great clinical significance, such as hematopoietic stem
cells or NRBCs.
* * * * *