U.S. patent application number 09/871645 was filed with the patent office on 2001-11-01 for cell separation method.
This patent application is currently assigned to Asahi Medical Co., Ltd.. Invention is credited to Sumita, Masaya, Terashima, Shuji.
Application Number | 20010036624 09/871645 |
Document ID | / |
Family ID | 27284685 |
Filed Date | 2001-11-01 |
United States Patent
Application |
20010036624 |
Kind Code |
A1 |
Sumita, Masaya ; et
al. |
November 1, 2001 |
Cell separation method
Abstract
A cell separation method comprising steps of introducing a
cell-containing fluid containing cells to be recovered and cells to
be removed, into a cell-capturing means capable of substantially
capturing the cells to be recovered and substantially permitting
passage therethrough of cells to be removed; taking out the
resulting fluid containing the cells to be removed, from the
cell-capturing means; and then introducing a liquid with a
viscosity of not more than 500 mPa.multidot.s and not less than 5
mPa.multidot.s into the cell-capturing means to recover therefrom
the cells to be recovered which have been captured by the
cell-capturing means.
Inventors: |
Sumita, Masaya; (Oita,
JP) ; Terashima, Shuji; (Oita, JP) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET 2ND FLOOR
ARLINGTON
VA
22202
|
Assignee: |
Asahi Medical Co., Ltd.
Tokyo
JP
J
|
Family ID: |
27284685 |
Appl. No.: |
09/871645 |
Filed: |
June 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09871645 |
Jun 4, 2001 |
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09341879 |
Jul 19, 1999 |
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6268119 |
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09341879 |
Jul 19, 1999 |
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PCT/JP98/00244 |
Jan 22, 1998 |
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Current U.S.
Class: |
435/2 |
Current CPC
Class: |
A61K 35/12 20130101;
A61M 2202/0439 20130101; A61M 1/3633 20130101; C12N 5/0087
20130101; C12N 5/0692 20130101 |
Class at
Publication: |
435/2 |
International
Class: |
C12N 005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 24, 1997 |
JP |
09-024517 |
Feb 24, 1997 |
JP |
09-054300 |
May 19, 1997 |
JP |
09-143002 |
Claims
We claim:
1. A liquid containing hematopoietic stem cells which is
substantially free from erythrocytes and platelets, and which has a
viscosity of not more than 500 mPa.multidot.s and not less than 5
mPa.multidot.s.
2. A cell-containing fluid according to claim 1, which contains at
least one cryopreservative agent.
3. A cell-containing fluid according to claim 2, wherein the
cryopreservative agent is one of an extracellular cryoprotectant
and an intracellular cryoprotectant.
4. A cell-containing fluid according to claim 1, which contains
dextran.
5. A liquid containing cells to be recovered and substantially no
cells to be removed which is obtained by a cell separation method
comprising steps of introducing a cell-containing fluid containing
cells to be recovered and cells to be removed into a cell-capturing
means capable of substantially capturing said cells to be recovered
and substantially permitting passage therethrough of said cells to
be removed; taking out the resulting fluid containing the cells to
be removed from said cell-capturing means; and then introducing a
liquid with a viscosity of not more than 500 mPa.multidot.s and not
less than 5 mPa.multidot.s into said cell-capturing means to
recover therefrom said cells to be recovered which have been
captured by said cell-capturing means.
6. A liquid for recovering captured cells from a cell-capturing
means which has a viscosity of not more than 500 mpa.multidot.s and
not less than 5 mPa.multidot.s.
7. A liquid for recovery according to claim 6, which can be used
also as a preservative for the cells.
8. A liquid for recovery according to claim 7, wherein the
preservation of the cells is cryopreservation.
9. A liquid for recovery according to claim 6, which is a solution
containing dextran.
10. A liquid for recovery according to claim 6, wherein the cells
are a mononuclear cell fraction containing hematopoietic stem
cells.
11. A liquid for recovery according to claim 6, wherein the cells
are hematopoietic stem cells.
Description
TECHNICAL FIELD
[0001] This invention relates to a method for separating and
recovering only necessary cells from a fluid containing a mixture
of various cells. The cells thus obtained can be used in providing
therapy for various diseases, such as hematopoietic stem cell
transplantation, and in fundamental sciences such as immunology and
cell biology.
BACKGROUND ART
[0002] Japanese patent JP-A-54-119012 discloses a technique for
recovering lymphocytes by capturing leukocytes on a filter from a
body fluid such as blood containing leukocytes (granulocytes,
monocytes and lymphocytes) and erythrocytes.
[0003] In the case of hematopoietic stem cell transplantation, cord
blood stem cells are noted as a source of hematopoietic stem cells
which does not cause any invasion to donors, and their clinical
application is vigorously attempted, mainly in countries in Europe
and America. Since cord blood stem cells are rarely transplanted to
a patient immediately after being collected from a donor, unlike in
other hematopoietic stem cell transfers, i.e., bone marrow
transplantation and peripheral blood stem cell transplantation,
they should be preserved for use after the collection. Such
preservation is often needed, particularly in the case of unrelated
setting. Before cryopreservation of cord blood, the separation of
nucleated cells and the removal of erythrocytes is considered
necessary in order to prevent side effects of erythrocytes lysis
after thawing, and to reduce the volume during the
cryopreservation. At present, cord blood is preserved after the
separation, in most cases ("Peripheral Blood Stem Cell
Transplantation" p. 173, NANKODO Ltd.). JP-B-8-69 discloses details
of a protocol for separating cord blood by a Ficoll-Hypaque method,
a centrifugation method using a liquid having an adjusted specific
gravity, hereinafter referred to as "Ficoll method". The Ficoll
method, however, is disadvantageous in that it is only feasible on
a laboratory level and requires very troublesome and time-consuming
operations. International Publication No. WO 96/17514 discloses a
bag system and method for separating erythrocytes in cord blood by
agglutination and precipitation by the use of hydroxyethyl starch
to obtain a concentrated nucleated cell suspension, and a cell
suspension obtained by that method. This method is somewhat
superior to the Ficoll method, a conventional method in that it
involves fewer troublesome operations, but it also is
time-consuming because two centrifugation runs are necessary.
[0004] On the other hand, some methods for separating hematopoietic
stem cells have been reported as substitutes for the Ficoll method
and the erythrocyte aggutination and removal. JP-A-8-104643
discloses a method for recovering hematopoietic stem cells by
capturing them on a filter permeable to erythrocytes, and then
causing a liquid flow in a direction opposite to the first liquid
flow direction. This method, however, merely uses Hanks' Balanced
Salt Solution (HBSS) as the liquid for the recovery.
[0005] Dextran is a polysaccharide composed of glucose units as
monomer units mainly by .alpha.-1,6 linkages, and has been used
since early times as an agent for separating leukocytes. The
separation of leukocytes by the use of dextran, however, utilizes
the effect of dextran as a hemagglutinating agent. After
erythrocytes in a test tube are agglutinated and precipitated,
centrifugation is carried out if necessary, and then leukocytes in
the supernatant are recovered with a pipet (Shiro Miwa, Rinsho
Kensa Gijutsu Zensho, Vol. 3, "Ketsueki Kensa" p. 425). Such an
effect is not characteristic of only dextran, because hydroxyethyl
starch and the like have the same hemagglutinating effect as that
of dextran.
[0006] Next, systems for separating hematopoietic stem cells are
described below. JP-A-7-184991 discloses an assembly for collecting
cord blood, in particular, a filter for removing contaminants in
cord blood, such as aggregates (e.g. micro-aggregates), tissue
particles, bone particles, steatomas, etc., which is provided
before a container for blood collection. This filter, however, is
not for capturing cells which should be recovered, but for removing
contaminants. Even if a material capable of capturing hematopoietic
stem cells is used in the filter by chance, this reference does not
describe the recovery of the captured hematopoietic stem cells at
all.
[0007] JP-A-8-52206 discloses an apparatus comprising a membrane
type plasma separator, as an apparatus for collecting cord blood
which is used for separating hematopoietic stem cells from cord
blood collected. This reference also discloses another separation
method using an apparatus for density gradient separation, i.e.,
separation by the Ficoll method.
[0008] The present invention is intended to provide a method for
separating cells which are desired to be recovered (hereinafter
referred to as "cells to be recovered" or "necessary cells") from a
mixture of necessary cells and unnecessary cells (hereinafter
referred to as "cells to be removed") by a simple and rapid
procedure. This procedure comprises a cell separation method which
captures necessary cells by use of a capturing means such as
filtering a fluid containing the cell mixture, and then recovering
the captured cells with high recovery. The present invention also
provides a line system obtained by embodiment of this method for
practical clinical employment. The present invention also provides
a recovering liquid used in said system, and a cell-containing
fluid obtained by using the method.
[0009] In order to solve the problems identified in the prior art,
the present inventors noted properties of a liquid for recovering
cells from a cell-capturing means, and earnestly investigated these
properties to conclude that when cells are recovered by using a
recovering liquid having a definite viscosity, a high recovery can
be attained. As a result of earnest investigation on the
compositions of various recovering liquids, the present inventors
found such a striking effect that, when cells are recovered by
using a physiological solution containing dextran, a very high
recovery can be attained. Thus, the objectives of the present
invention have been accomplished.
DISCLOSURE OF THE INVENTION
[0010] One aspect of the present invention is directed to a cell
separation method comprising steps of introducing a cell-containing
fluid containing cells to be recovered and cells to be removed into
a cell-capturing means capable of substantially capturing the cells
to be recovered and substantially permitting passage therethrough
of the cells to be removed. Then, the resulting fluid containing
the cells to be removed is taken from the cell-capturing means, and
then a liquid with a viscosity of not more than 500 mPa.multidot.s
and not less than 5 mPa.multidot.s is introduced into the
cell-capturing means to recover therefrom the cells to be recovered
which have been captured by the cell-capturing means.
[0011] Another aspect of the present invention is directed to a
cell separation and preservation method comprising steps of
introducing a cell-containing fluid containing cells to be
recovered and cells to be removed, into a cell-capturing means
capable of substantially capturing the cells to be recovered, and
substantially permitting passage therethrough of the cells to be
removed. The resulting fluid containing the cells to be removed is
taken out of the cell-capturing means, and a liquid with a
viscosity of not more than 500 mPa.multidot.s and not less than 5
mPa.multidot.s is introduced into the cell-capturing means to
recover therefrom the cells to be recovered which have been
captured by the cell-capturing means. The recovered cells are then
preserved.
[0012] Another aspect of the present invention is directed to a
cell separation and preservation method comprising steps of
introducing a cell-containing fluid containing cells to be
recovered and cells to be removed into a cell-capturing means
capable of substantially capturing the cells to be recovered, and
substantially permitting passage of the cells to be removed. The
resulting fluid containing the cells to be removed is taken from
the cell-capturing means, and a liquid with a viscosity of not more
than 500 mPa.multidot.s and not less than 5 mPa.multidot.s is
introduced into the cell-capturing means to recover therefrom the
cells to be recovered which have been captured by the
cell-capturing means. The recovered cells are then subjected to
cryopreservation and thawing.
[0013] Still another aspect of the present invention is directed to
a cell separation system comprising a cell-capturing means which is
capable of substantially capturing cells to be recovered and
substantially permitting passage therethrough of cells to be
removed, which has at least an inlet and an outlet. A line for
introducing into the cell-capturing means a cell-containing fluid
containing the cells to be recovered and the cells to be removed is
connected upstream to the inlet of the cell-capturing means. A line
for introducing a liquid into the cell-capturing means is connected
downstream to the outlet of the cell-capturing means, and a line
for cell recovery from the inlet side of the cell-capturing means
is connected upstream to the inlet of the cell-capturing means.
[0014] Still another aspect of the present invention is directed to
a cell separation method comprising steps of introducing a
cell-containing fluid containing cells to be recovered and cells to
be removed into a cell-capturing means capable of substantially
capturing the cells to be recovered and substantially permitting
passage therethrough of the cells to be removed, through a line
connected upstream to the inlet of the cell-capturing means. The
resulting fluid containing the cells to be removed is taken out
through the outlet of the cell-capturing means, and then a liquid
with a viscosity of not more than 500 mPa.multidot.s and not less
than 5 mPa.multidot.s is introduced into the cell-capturing means
through a line connected downstream to the outlet of the
cell-capturing means to recover the cells to be recovered which
have been captured by the cell-capturing means, through a line
connected upstream to the inlet of the cell-capturing means.
[0015] Still another aspect of the present invention is directed to
a liquid containing hematopoietic stem cells which is substantially
free from erythrocytes and/or platelets and has a viscosity of not
more than 500 mPa.multidot.s and not less than 5
mPa.multidot.s.
[0016] Still another aspect of the present invention is directed to
a liquid containing cells to be recovered and substantially having
no cells to be removed which is obtained by a cell separation
method comprising steps of introducing a cell-containing fluid
containing cells to be recovered and cells to be removed into a
cell-capturing means capable of substantially capturing the cells
to be recovered and substantially permitting passage therethrough
of the cells to be removed. The resulting fluid containing the
cells to be removed is taken out from the cell-capturing means, and
then a liquid with a viscosity of not more than 500 mPa.multidot.s
and not less than 5 mPa.multidot.s is introduced into the
cell-capturing means to recover therefrom the cells to be recovered
which have been captured by the cell-capturing means.
[0017] Still another aspect of the present invention is directed to
a liquid for recovering captured cells from a cell-capturing means
which has a viscosity of not more than 500 mPa.multidot.s and not
less than 5 mPa.multidot.s.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is one embodiment of the cell separation system
according to the present invention.
[0019] FIG. 2 is a schematic view of a cell separation system used
in Example 1.
[0020] FIG. 3 is a schematic view of a cell separation system used
in Example 4.
BEST MODE FOR CARRYING OUT THE INVENTION
[0021] In the present specification, the term "cells to be
recovered" means cells used for some purpose after their separation
and recovery. The term "cells to be removed" means cells
unnecessary for the above purpose or cells which should be
positively removed because they are, for example, pathogenic cells,
so that contamination by them of cells to be recovered causes a
problem.
[0022] The cell-containing fluid containing cells to be recovered
and cells to be removed can be but is not limited to peripheral
blood, bone marrow, cord blood (including not only that collected
through a umbilical cord blood vessel but also that collected
through a placenta blood vessel), lymph fluids, and those obtained
by subjecting the above fluids to some treatment such as
centrifugation, and suspensions obtained by resuspending cells
extracted from any of various organs or tissues, in some
liquid.
[0023] The term "nucleated cells" means cells having a nucleus
therein. The nucleated cells include, for example, leukocytes,
granulocytes, neutrophils, baso-phils, eosinophils, myelocytes,
erythroblasts, lymphocytes, T lymphocytes, helper T lymphocytes,
cytotoxic T lymphocytes, suppressor T lymphocytes, B lymphocytes,
NK cells, NKT cells, monocytes, macrophages, dendritic cells,
osteoclasts, osteoblasts, osteocytes, hematopoietic stem cells,
fibroblasts and chondroblasts.
[0024] The term "mononuclear cell fraction containing hematopoietic
stem cells" means a mononuclear cell population containing
hematopoietic stem cells and/or hematopoietic progenitor cells
(they are hereinafter given the general name "hematopoietic stem
cells"). "Mononuclear cell" is a general term for cells having a
nucleus therein, and specific examples thereof are lymphocytes (T
cells, B cells and NK cells), monocytes, hematopoietic stem cells,
myelocytes, blast cells, etc.
[0025] The content of hematopoietic stem cells in the mononuclear
cell population is usually 0.01% to 99% and varies depending on the
kind of a starting cell population, and whether cells are treated
or not. The content of hematopoietic stem cells is usually, for
example, about 0.01% in peripheral blood, 0.05 to 1.0% in cord
blood and 0.5 to 2% in bone marrow in the case of a normal person.
In peripheral blood having a granulocyte colony-stimulating factor
(G-CSF) administered, the content of hematopoietic stem cells
differs markedly among individuals, and is 0.1 to several per cent.
When cell separation using a monoclonal antibody, in particular,
cell separation by a flow cytometry method is carried out, the
content of hematopoietic stem cells reaches 99% in some cases. In
any case, the term "mononuclear cell fraction containing
hematopoietic stem cells" does not concretely specify the content
of hematopoietic stem cells at all.
[0026] The cells having no nucleus which are referred to in the
present specification include, for example, erythrocytes and
platelets.
[0027] The expression "cells to be removed have a surface marker
different from that of cells to be recovered" in the present
specification means that the cells to be recovered and the cells to
be removed are similarly nucleated cells, but are different in
surface marker (the cells to be recovered and the cells to be
removed belong different subgroups, respectively). For example, the
cells to be recovered are helper T lymphocytes (having anti-CD4
antigen as a surface marker), and the cells to be removed are
suppressor T lymphocytes (having anti-CD8 antigen as a surface
marker).
[0028] When cells to be recovered are nucleated cells, and cells to
be removed are cells having no nucleus, examples of their
combination and examples of use thereof are as follows, but the
combination and use are not limited thereto.
[0029] 1. Cells to be recovered: leukocytes, cells to be removed:
erythrocytes, use: interferon preparation.
[0030] 2. Cells to be recovered: lymphocytes, cells to be removed:
erythrocytes and platelets, use: adoptive-immuno therapy.
[0031] 3. Cells to be recovered: a mononuclear cell fraction
containing hematopoietic stem cells, cells to be removed:
erythrocytes and plate- lets, use: hematopoietic stem cell
transplantation.
[0032] When cells to be recovered are nucleated cells, and cells to
be removed are nucleated cells having a surface marker different
from that of the cells to be recovered, examples of their
combination and examples of use thereof are as follows, but the
combination and use are not limited thereto.
[0033] 1. Cells to be recovered: CD34-positive nucleated cells,
cells to be removed: CD34-negative nucleated cells, use:
CD34-positive cell transplantation.
[0034] 2. Cells to be recovered: CD8-positive T lymphocytes, cells
to be removed: CD8-negative T lymphocytes, use: adoptive-immuno
therapy.
[0035] When cells to be recovered are nucleated cells and cells to
be removed are cells having no nucleus and nucleated cells having a
surface marker different from that of the cells to be recovered,
examples of their combination and examples of use thereof are as
follows, but the combination and use are not limited thereto.
[0036] 1. Cells to be recovered: CD34-positive nucleated cells,
cells to be removed: erythrocytes, platelets and CD34-negative
nucleated cells, use: CD34-positive cell transplantation.
[0037] 2. Cells to be recovered: CD8-positive T lymphocytes, cells
to be removed: erythrocytes, platelets and CD8-negative T
lymphocytes, use: adoptive-immuno therapy.
[0038] In the present invention, the cell-capturing means capable
of capturing at least cells to be recovered and substantially
permitting passage therethrough of cells to be removed, may
comprise a container having a liquid inlet and a liquid outlet
which is packed with a material capable of capturing the cells to
be recovered and substantially permitting passage therethrough of
the cells to be removed, and a molded container having a
cell-capturing surface on its inner surface. The material capable
of capturing the cells to be recovered and substantially permitting
passage therethrough of the cells to be removed may be any
conventional cell-capturing material so long as it can selectively
capture the cells to be recovered. The following materials, for
example, are preferable because of their excellent moldability,
sterilizability, and low cytotoxicity: synthetic polymers such as
polyethylenes, polypropylenes, polystyrenes, acrylic resins,
nylons, polyesters, polycarbonates, polyacrylamides, polyurethanes,
etc.; natural polymers such as agarose, cellulose, cellulose
acetate, chitin, chitosan, alginates, etc.; inorganic materials
such as hydroxyapatite, glass, alumina, titania, etc.; and metals
such as stainless steel, titanium, aluminum, etc.
[0039] These capturing materials may be used as they are, or after
being subjected to surface modification necessary for selective
passage or capture of cells, etc. For example, for improving the
permeability to platelets, there is, for instance, the method
comprising coating with a polymer having nonionic hydrophilic
groups and basic nitrogen-containing functional groups which has
been proposed in International Publication No. WO 87/05812. As a
method for selective capture of cells, a method of immobilizing a
ligand having affinity for specific cells, such as an amino acid,
peptide, sugar or glycoprotein (including bio-ligands such as
antibody and adhesion molecules) may be used, for example, by the
haloaceamide method proposed in JP-A-2-261833.
[0040] The shape of the capturing material may be granular, a fiber
mass, woven fabric, nonwoven fabric, a spongy structure, a flat
plate, etc. The granules, fiber mass, woven fabric, nonwoven fabric
and spongy structure are preferable because they have a large
surface area per volume. From the viewpoint of ease of handling,
porous structures such as the fiber mass, woven fabric, nonwoven
fabric and spongy structure are more preferable. Among them, the
nonwoven fabric and spongy structure are the most preferable from
the viewpoint of the flowability of a cell suspension and
productivity.
[0041] When the nonwoven fabric is used, its fiber diameter in the
case of not immobilizing a so-called bio-ligand capable of
specifically binding to specific cells, such as anti-CD34
monoclonal antibody on the fabric surface, is usually not more than
30 .mu.m and not less than 1.0 .mu.m, preferably not more than 20
.mu.m and not less than 1.0 .mu.m, more preferably not more than 10
.mu.m and not less than 1.5 .mu.m. When the fiber diameter is less
than 1.0 .mu.m, the cells to be recovered are undesirably liable to
be tightly captured and become difficult to recover. When the fiber
diameter is more than 30 .mu.m, the cells to be recovered are very
likely to pass through the nonwoven material without being captured
by fiber. Both of such values are not desirable because they tend
to decrease the recovery.
[0042] When the spongy structure is used, its pore size is usually
not more than 25 .mu.m and not less than 2.0 .mu.m, preferably not
more than 20 .mu.m and not less than 3.0 .mu.m, more preferably not
more than 15 .mu.m and not less than 4.0 .mu.m. When the pore size
is less than 2.0 .mu.m, the flowability is very low, so that the
passage of a fluid through the spongy structure tends to be
difficult in itself. When the pore size is more than 25 .mu.m, the
capture rate of the cells to be recovered is undesirably decreased,
resulting in a low recovery.
[0043] The container packed with material capable of capturing the
cells to be recovered and substantially permitting passage
therethrough of the cells to be removed preferably uses, but is not
limited to the following materials, for example, because of their
excellent moldability, sterilizability, and low cytotoxicity:
synthetic polymers such as polyethylenes, polypropylenes,
polystyrenes, acrylic resins, nylons, polyesters, polycarbonates,
polyacrylamides, polyurethanes, poly(vinyl chloride)s, etc.;
inorganic materials such as hydroxyapatite, glass, alumina,
titania, etc.; and metals such as stainless steel, titanium,
aluminum, etc.
[0044] The molded container having a cell-capturing surface on its
inner surface, i.e., an example of the cell-capturing means which
is other than the container packed with the cell-capturing
material, may be a flask, dish, conical tube, syringe, blood bag,
etc.
[0045] In the present specification, the expression "substantially
capturing cells to be recovered" means capturing 60% or more of
cells to be recovered in a cell-containing fluid. The expression
"substantially permitting passage therethrough of cells to be
removed" means passing 60% or more of cells to be removed in the
cell-containing fluid.
[0046] In the present invention, cells to be recovered which have
been captured by the cell-capturing means are recovered by using a
liquid with a specific viscosity (hereinafter referred to also as
"recovering liquid" or "liquid for recovery"). The viscosity of
this liquid should not be more than 500 mPa.multidot.s and not less
than 5 mPa.multidot.s, preferably not more than 100 mPa.multidot.s
and not less than 5 mPa.multidot.s, more preferably not more than
50 mPa.multidot.s and not less than 7 mPa.multidot.s. When the
viscosity is less than 5 mPa.multidot.s, the recovery is low. When
the viscosity is more than 500 mPa.multidot.s, the passage of the
liquid through the cell-capturing means is very difficult even if a
pump is used, so that the work-efficiency is low. Moreover, a
pressure increase is caused, so that leakage from a joint between
tubes in a line tends to occur. Therefore, such viscosity values
are not desirable. As a method for measuring the viscosity, use of
a rotating viscometer is preferable because it is the simplest, and
has a high precision.
[0047] Any liquid may be used as the recovering liquid, so long as
it has little undesirable influence on cells. For example,
solutions of synthetic polymers such as poly(ethylene glycol)s
poly(vinylpyrrolidone)s- , poly(vinyl alcohol)s etc.; solutions of
natural polymers such as methyl cellulose, gelatin, hydroxyethyl
starch, dextran, chitin derivatives, collagen, fibronectin,
albumin, globulin, etc.; solutions of organic substances such as
glucose, saccharose, maltose, trehalose, sorbitol, glycerol,
dimethyl sulfoxide, silicone oil, etc.; and mixtures thereof may be
used. As a result of investigation by the present inventors, it was
found that an especially high recovery can be attained by using
dextran. Therefore, employment of dextran is explained below in
detail.
[0048] The dextran referred to herein is a glucose polymer in which
most of the glucose units are joined by .alpha.-1,6 linkages. The
dextran includes its partial hydrolysis products and its
derivatives such as sulfate esters. Although the dextran is not
limited in molecular weight, its average molecular weight is
preferably 1,000 to 10,000,000, more preferably 5,000 to 5,000,000,
most preferably 10,000 to 1,000,000, in view of solubility,
availability, etc. Since the viscosity varies depending on the
molecular weight, even at the same concentration, the molecular
weight of the concentration is properly adjusted so that the
viscosity may be not more than 500 mPa.multidot.s and not less than
5 mPa.multidot.s. A sterilized dextran 40 injection (a 10 w/v%
solution of dextran with a molecular weight of about 40,000 in
physiological saline), approved as a medicine, is on the market and
hence can be suitably used. In order to adjust the viscosity to not
more than 500 mPa.multidot.s and not less than 5 mPa.multidot.s,
the dextran may be used singly, or in admixture with other
substances. Examples of the substances are synthetic polymers such
as poly(ethylene glycol)s, poly(vinyl-pyrrolidone)s, poly(vinyl
alcohol)s, etc.; natural polymers such as methyl cellulose,
gelatin, hydroxyethyl starch, dextran, chitin derivatives,
collagen, fibronectin, albumin, globulin, etc.; and organic
substances such as glucose, saccharose, maltose, trehalose,
sorbitol, glycerol, dimethyl sulfoxide, etc. Although a mechanism
by which cells can be recovered with high recovery by using dextran
is not known at present, the present inventors conjecture that the
dextran has a property of reducing the adhesiveness of the cells to
the capturing material.
[0049] The solvent used for dissolving a solute in the preparation
of the liquid having a viscosity of not more than 500
mPa.multidot.s and not less than 5 mPa.multidot.s, may be
physiological saline, buffer solutions such as Dulbecco phosphate
buffer solution (D-PBS), Hank's Balanced Salt Solution (HBSS) and
the like, and media such as RPMI1640 and the like. If necessary,
dextran, hydroxyethyl starch, albumin, globulin, glucose,
saccharose, trehalose, globulin, citrate-phosphate-dextrose (CPD),
acid-citrate-dextrose (ACD), EDTA, heparin, etc. may be
incorporated into the liquid for supply of a nutriment, protection
of cell membrane, or impartment of anticoagulating effect, etc.
[0050] The liquid with a specific viscosity according to the
present invention is preferably one which can be used for
cryopreservation of cells to be recovered, or preservation of the
cells in a liquid state. As described above, for hematopoietic stem
cell transplanation, in particular, hematopoietic stem cell
transplanation using cord blood, a cell population freed of
erythrocytes by a Ficoll method or the like is washed (because a
Ficoll solution is toxic), and a cryoprotectant and the like are
added thereto to prepare a cell suspension, followed by
cryopreservation in liquid nitrogen or a freezer until needed for
practical use. In the present invention, a cell suspension to be
preserved can be prepared without troublesome operations after cell
separation by using a liquid suitable both for the preservation, in
particular, cryopreservation, as well as for recovery, by having a
specific viscosity. Specific examples of the liquid for recovery
which is usable for cryopreservation and as a cryoprotectant are, a
nutriment, or a cell membrane protecting component, etc.
Cryoprotectants are classified into two categories, 1)
extracellular cryoprotectants, and 2) intracellular
cryoprotectants, according to the action mechanism. In the first
category, water-soluble polymers such as hydroxyethyl starch,
dextran, poly(vinylpyrrolidone)s, etc. are generally used. In the
second category, low-molecular weight organic compounds such as
dimethyl sulfoxide, glycerol, etc. are generally used. The
nutriment includes sugars such as glucose and the like, and various
media for cell culture. As the cell membrane protecting component,
albumin is generally used. Plasma is used in some cases as a
combination of the nutriment and the cell membrane protecting
component. As described above, these components are preferably used
singly, or in combination in the liquid for recovery having a
specific viscosity of the present invention. The components
described above may be added at the time of cryopreservation after
cell recovery.
[0051] There are generally two freezing methods employed, i.e., a
simple method using a deep-freezer at -80.degree. C., or a method
comprising slow cooling in a program freezer and preservation in
liquid nitrogen. For thawing cells subjected to cryopreservation,
rapid thawing in a warm bath at 37.degree. C. is generally carried
out.
[0052] As a method for introducing the cell-containing fluid
referred to in the present specification into the cell-capturing
means, there may be adopted either a method of connecting a bag or
bottle containing the cell-containing fluid through a tube, and
then introducing the fluid, for example, by utilizing its fall, a
roller pump, causing a flow of the fluid by squeezing the bag, or
by a method of connecting a syringe containing the cell-containing
fluid, and introducing the fluid by pushing the piston of the
syringe by hand or using a device such as a syringe pump. The
pushing by hand is characterized by its simplicity, and the use of
the device is characterized in that the control of the flow rate of
the recovering liquid in its introduction is easy. Therefore, a
suitable method is selected depending on the purpose.
[0053] When the cell-containing fluid is introduced into the
cell-capturing means, the cells to be recovered are captured, and
the cells to be removed flow out, but a minority thereof remain in
the container in some cases. Therefore, the cell-capturing means is
preferably rinsed in order to rinse away the slight amount of the
remaining cells to be removed. Any rinse may be used, so long as it
is a physiological solution. Several examples thereof are
physiological saline, buffer solutions such as Dulbecco phosphate
buffer solution (D-PBS), Hank's Balanced Salt Solution (HBSS) and
the like, and media such as RPMI1640 and the like. If necessary,
dextran, hydroxyethyl starch, albumin, globulin, glucose,
saccharose, trehalose, globulin, citrate-phosphate-dextrose (CPD),
acid-citrate-dextrose (ACD), EDTA, or heparin, etc. may be added to
the physiological solutions mentioned above for supply of a
nutriment, protection of cell membrane, and impartment of
anticoagulating effect, etc.
[0054] There are two directions for introduction of the rinse,
i.e., the same direction as the direction of introduction of the
cell-containing fluid, and the direction opposite thereto. Of
these, the same direction is preferable. In the case of the
opposite direction, the cells to be recovered which have been
captured are liable to leak out owing to the rinsing. The viscosity
of the rinse is preferably less than 5 mPa.multidot.s. When the
viscosity is 5 mPa.multidot.s or more, the cells to be recovered
which have been captured are liable to leak out.
[0055] In the present invention, as a method for introducing the
liquid with a viscosity of not more than 500 mPa.multidot.s and not
less than 5 mPa.multidot.s into the above-mentioned cell-capturing
means, there may be adopted either a method of connecting a bag or
bottle containing the liquid to the cell-capturing means through a
tube, and introducing the liquid by utilizing its fall, a roller
pump, by squeezing the bag, or by a method of connecting a syringe
containing the liquid, and introducing the liquid into the
cell-capturing means by pushing the piston of the syringe by hand,
or by using a device such as a syringe pump. In this case, as in
the direction of introduction of the liquid, there are two
directions, i.e., the same direction as the direction of
introduction of the cell-containing fluid, and the direction
opposite thereto. Of these, the latter is usually preferable
because the cell recovery is higher. The flow rate of the
recovering liquid is preferably rapid because the recovery tends to
be increased. The linear speed obtained by dividing the flow rate
by the filtration sectional area is usually 0.5 cm/min. or more,
preferably 5 cm/min. or more, and more preferably 10 cm/min. or
more.
[0056] It is also possible to recover a slight amount of cells (or
their constituents) remaining in the cell-capturing means, by
introducing another liquid after introducing the recovering liquid.
By this recovery, the collection of a sample for HLA typing, which
is indispensable, for example, in hematopoietic stem cell
transplantation, can be carried out simultaneously with the cell
separation procedure. A slight amount of the cells (or their
constituents) remaining in the cell-capturing means are used for
various purposes, other than HLA typing such as investigation of ex
vivo expansion of hematopoietic stem cells, genetic diagnosis, or
employment in cell transplantation in combination with the cells
obtained by the first recovery. A brief supplementary explanation
of HLA typing is given below.
[0057] HLA typing is carried out by using DNA present in the nuclei
of nucleated cells. Therefore, recovering the DNA is preferable to
recovering the cells themselves because it is laborsaving.
Accordingly, a liquid capable of lysing or disrupting the cells is
preferably used as a recovering liquid. The liquid includes, for
example, hypotonic liquids such as solutions of surfactants (e.g.
sodium dodecyl sulfate, lauryl sodium sulfate and Triton X-100),
distilled water, ion-exchanged water, etc. The DNA recovered by the
use of such a liquid is purified by a well-known phenol chloroform
method or the like and subjected to HLA typing.
[0058] In the present invention, the recovered cells may be
preserved until use. For the preservation, there are two methods,
preservation in a liquid state, and cryopreservation. The
cryopreservation is usually carried out because the preservation in
a liquid state is limited in time to at most 2 to 3 days in the
case of, for example, hematopoietic stem cells.
[0059] Next, the cell separation system of the present invention is
explained below. The line referred to in the present specification,
i.e., the line for introducing the cell-containing fluid into the
cell-capturing means which is connected upstream to the inlet of
the cell-capturing means is a line connectable to, for example, a
container reserving the cell-containing fluid, or a line
connectable to a living body tissue in which the cell-containing
fluid is present. Specific examples of the former are as follows: a
tube equipped with a spike or a tube equipped with a Luer adapter
(male or female) is properly selected when the container reserving
the cell-containing fluid is a blood bag, or a mere tube is
properly selected when connection by a sterilized connector
(hereinafter referred to as "SCD connection") is made. In addition,
a needlable tube having a septum is properly selected as the line
when the container reserving the cell-containing fluid is a syringe
equipped with a needle, or a Luer adapter (female) is properly
selected as the line when the container is a syringe having a Luer
opening but not a needle. Specific examples of the latter line are
as follows, for example, when cord blood is used, the aforesaid
living body tissue is umbilical cord and/or placenta, and a tube
equipped with a metallic needle stickable into them is mentioned as
the latter line. When a tube is used, it may be equipped between
its ends with a clamp for opening or shutting the line, a roller
clamp for adjusting the flow rate, a mesh chamber for removing
aggregates, a syringe for giving the flow rate (including a flow
path changing means), etc. When a syringe is used, it may be
directly connected to the inlet of the cell-capturing means without
a tube.
[0060] The other line referred to in the present specification,
i.e., the line for introducing a liquid into the aforesaid
cell-capturing means which is connected downstream to the outlet of
the aforesaid cell-capturing means, includes lines which are
classified as follows according to whether a container containing
the liquid to be introduced into the cell-capturing means has been
previously connected or is subsequently connectable, and according
to the means used for introducing the liquid. That is, when the
container containing the liquid to be introduced into the
cell-capturing means is previously connected, the line includes,
for example, a tube equipped with a bag, and a syringe. In the case
of such a bag, a method for introducing the liquid into the
cell-capturing means includes a method utilizing the fall of the
liquid, a method of squeezing the bag, a method using a roller
pump, etc. When the container containing the liquid to be
introduced into the cell-capturing means is connected afterwards,
the following tubes are selected. When a syringe is used, the line
includes a needlable tube having a septum, a tube equipped with a
Luer adapter (female), a tube equipped with a three-way stopcock,
etc., to which the syringe can be connected. When a bag is used, a
line connectable to the bag, i.e., a tube equipped with a spike, or
a tube equipped with a Luer adapter (male or female) is properly
selected as the aforesaid line. When SCD connection is made, a mere
tube is properly selected as the aforesaid line. When a syringe is
used, it may be directly connected to the outlet of the
cell-capturing means without a tube.
[0061] The other line referred to in the present specification,
i.e., the line for recovering cells from the inlet side of the
aforesaid cell-capturing means which is connected upstream to the
inlet of the aforesaid cell-capturing means, includes lines which
are classified as follows according to a container for recovering
cells which flow out of the cell-capturing means. That is, when the
cells are recovered into a bag, a line connected or connectable to
the bag, i.e., a tube equipped with a spike or a tube equipped with
a Luer adapter (male or female) is properly selected as the
aforesaid line. When SCD connection is made, a mere tube is
properly selected as the aforesaid line. When the cells are
collected into a conical tube, any open-ended line may be used.
When the cells are collected by using a syringe having a Luer
opening, a Luer adapter (female), a three-way stopcock and the like
are used. When a syringe is used, it may be directly connected to
the inlet of the cell-capturing means without a tube.
[0062] Instead of this other line, for example, a container for
recovering the cells which flow out of the cell-capturing means is
preferably able to withstand freezing and thawing, such as a freeze
bag, because the transfer of the cells to a freeze bag can then be
omitted. Examples of cryopreservation bags are freeze bags such as
"Cryocyte" manufactured by Baxter, "Cell Freeze Bag" manufactured
by Charter Med, "Hemo Freeze Bag" manufactured by NPBI, etc.
[0063] To the cell separation system according to the present
invention, a line for introducing a liquid into the cell-capturing
means may be added in order to rinse away a slight amount of cells
to be removed which remain in the cell-capturing means, before
recovering cells captured by the cell-capturing means. This line
includes lines which are classified as follows according to whether
a container containing the liquid is previously connected, or
subsequently connectable, and according to the means for
introducing the liquid. That is, when the container containing the
liquid is previously connected, the line includes, for example, a
tube equipped with a bag, and a syringe. When the container
containing the liquid is connected afterwards, the following types
of tubes are selected. When a syringe is used, the line includes a
needlable tube having a septum, and a tube equipped with a Luer
adapter (female), to which the syringe can be connected. When a bag
is used, a line connectable to the bag, i.e., a tube equipped with
a spike or a tube equipped with a Luer adapter (male or female) is
properly selected as the line. When an SCD connection is made, a
mere tube is properly selected as said line. When a syringe is
used, it may be directly connected to the outlet of the
cell-capturing means without a tube. Although the position of
connecting said line to the cell-capturing means may be on either
the inlet side or the outlet side, it is preferably on the inlet
side from the viewpoint of ease of operation.
[0064] The present cell separation system, may have a line added
for collecting cells (or their constituents) remaining in the
cell-capturing means by further introducing a liquid after
recovering cells to be recovered. In the case where cells different
in purpose of use from the first recovered cells are recovered, for
example, in the case where a solution capable of lysing or
disrupting cells is used for collecting cells (or their
constituents) remaining in the cell-capturing means for HLA typing,
the line should comprise a means for changing the flow path, and a
plurality of branches so that the cells (or their constituents)
collected afterward will not be mixed with the first recovered
cells. The flow path changing means may include clamps, spikes,
etc.
[0065] The cell separation method using the above-mentioned line
system comprises steps of introducing, through a line connected
upstream, a cell-containing fluid containing cells to be recovered
and cells to be removed into a cell-capturing means capable of
substantially capturing the cells to be recovered and substantially
permitting passage of the cells to be removed. The resulting fluid
containing the cells to be removed is taken out through the outlet
of the cell-capturing means, and then a liquid with a viscosity of
not more than 500 mPa.multidot.s and not less than 5 mPa.multidot.s
is introduced into the cell-capturing means through a line
connected downstream from the outlet of the cell-capturing means in
order to recover the cells. When the recovered cells are preserved,
the line (e.g. a freeze bag) connected upstream to the inlet of the
cell-capturing means and containing the cells recovered, is sealed
up and separated. The sealing-up and separation are carried out,
for example, as follows: the line is sealed up by heat fusion using
a heat sealer or the like, and then cut off, or a tube connected
through a Luer adapter is detached from the main body and then
heat-fused by using a heat sealer or the like. In any case, the
term "sealing-up and separation" does not specify the order of
operations (e.g. sealing-up followed by separation) at all.
[0066] The present invention further provides a liquid which
contains hematopoietic stem cells which is substantially free from
erythrocytes and/or platelets, and has a viscosity of not more than
500 mPa.multidot.s and not less than 5 mPa.multidot.s. The
expression "substantially free from" used here means that this
cell-containing fluid is prepared by removing 60% or more of
erythrocytes and/or platelets from a starting cell-containing
fluid. Although cord blood contains erythrocytes in addition to
hematopoietic stem cells, a hematopoietic stem cell suspension
containing substantially no erythrocyte can be provided by
employing the cell separation method of the present invention.
Furthermore, the cell-containing fluid may contain a
cryopreservative agent.
[0067] The present invention still further provides a liquid
containing cells to be recovered and substantially no cells to be
removed which is obtained by a cell separation method comprising
steps of introducing a cell-containing fluid containing cells to be
recovered and cells to be removed, into a cell-capturing means
capable of substantially capturing said cells to be recovered and
substantially permitting passage therethrough of said cells to be
removed. The resulting fluid containing the cells to be removed is
taken out of the cell-capturing means, and then a liquid with a
viscosity of not more than 500 mPa.multidot.s and not less than 5
mPa.multidot.s is introduced into the cell-capturing means to
recover the cells which have been captured by the cell-capturing
means. When the separation method of the present invention is
applied to a suspension containing cells to be recovered and cells
to be removed, it becomes possible to efficiently provide a
suspension substantially comprising the cells to be recovered.
[0068] The present invention still further provides a liquid with a
viscosity of not more than 500 mPa.multidot.s and not less than 5
mPa.multidot.s as a liquid for recovering captured cells from a
cell-capturing means. This liquid is preferably one which can be
used also as a preservative for cells. In the case of preservation
in a liquid state, specific examples of the preservative are sugars
(e.g. glucose), nutriments (e.g. various media for cell culture),
cell membrane protecting components (e.g. albumin), and
combinations of a nutrient and a cell membrane protecting component
(e.g. plasma). In the case of cryopreservation, the preservative
includes cryoprotectants, in addition to the above examples. The
cryoprotectants are classified into two categories, 1)
extracellular cryoprotectants, and 2) intracellular
cryoprotectants, according to the action mechanism. In the first
category, water-soluble polymers such as hydroxyethyl starch,
dextran, and poly(vinylpyrrolidone)s, etc. are generally used. In
the second category, low-molecular weight organic compounds such as
dimethyl sulfoxide, and glycerol, etc. are generally used.
[0069] An embodiment of the cell separation system according to the
present invention is explained below with reference to the
drawings, which should not be construed as limiting the scope of
the invention.
[0070] FIG. 1 shows one embodiment of the cell separation system
according to the present invention. In this system, all of the
following connections are made by the use of spikes: the connection
of a starting-cell bag (containing a cell-containing fluid
containing cells to be recovered and cells to be removed) to the
main body of the system of the present invention; the connection of
a bag for recovering a fluid which flows out through the outlet of
a cell-capturing means, to the main body of the system of the
present invention; and the connection of a bag for recovering cells
from the outlet side of the cell-capturing means, to the main body
of the system of the present invention. In the system, there is a
three-way stopcock provided to which a syringe with a male Luer
opening is connected for introducing a liquid into the
cell-capturing means.
[0071] In FIG. 1, numeral 1 denotes the cell-capturing means
capable of substantially capturing the cells to be recovered and
substantially permitting passage therethrough of the cells to be
removed. Numeral 2 denotes a line for introducing the
cell-containing fluid into the cell-capturing means from the
starting-cell bag, which comprises a spike 2-1, a clamp 2-2 and a
tube 2-3. Numeral 3 denotes a line for discharging the fluid which
flows out through the outlet of the cell-capturing means 1, which
comprises a spike 3-1 and a tube 3-2. Numeral 4 denotes a line for
introducing the liquid into the cell-capturing means from the
outlet side of the cell-capturing means 1, which shares the tube
with the line 3 and has the three-way stopcock 4-1 to which the
syringe is connected. Numeral 5 denotes a line for recovering cells
from the inlet side of the cell-capturing means, which comprises a
spike 5-1, clamp 5-2, a tube 5-3 and a part of the tube 2-3. This
line shares the tube 2-3 with the line 2 from the inlet of the
cell-capturing means 1 to the point at which the tube 5-3 diverges
from the tube 2-3.
[0072] Next, a method for using the cell-capturing means is
explained below. Initially, the clamp 2-2 is shut, the three-way
stopcock 4-1 is closed only in the direction of syringe connection,
and the clamp 5-2 is closed. Then, the spike 2-1 is stuck into the
starting-cell bag and the spike 3-1 is stuck into an empty bag.
When the clamp 2-2 is opened, the cell-containing fluid is supplied
to the cell-capturing means 1 through the tube 2-3 of the line 2.
The cells to be recovered are captured and the cells to be removed
are taken out and then collected in the empty bag through the tube
3-2 of the line 3. After completion of the treatment of the
cell-containing fluid, the clamp 2-2 is closed, and the spike 2-1
is pulled out of the starting-cell bag and stuck into a
commercially available bottle of physiological saline. When the
clamp 2-2 is opened, the physiological saline rinses the
cell-capturing means 1 and is collected in the bag containing the
collected cells to be removed, through the line 3. After completion
of the rinsing, the clamp 2-2 and the tube 3-2 are closed.
Subsequently, a syringe containing a liquid with a viscosity of not
more than 500 mPa.multidot.s and not less than 5 mPa.multidot.s is
connected to the three-way stopcock 4-1, and the spike 5-1 is stuck
into a cell-recovering bag. The three-way stopcock is turned in
such a direction that the syringe communicates only with the
cell-capturing means 1. After the clamp 5-2 is opened, the piston
of the syringe is pushed to introduce the liquid into the
cell-capturing means 1 from its outlet side, whereby the cells
captured by the cell-capturing means are recovered into the
cell-recovering bag through the line 5.
[0073] The present invention is illustrated below in further detail
with reference to examples, which should not be construed as
limiting the scope of the invention.
EXAMPLE 1
[0074] This working example shows an example of cell separation in
the case where a cell-containing fluid was cord blood, cells to be
recovered are a mononuclear cell fraction containing hematopoietic
stem cells, and cells to be removed are erythrocytes and
platelets.
[0075] {circle over (1)} Cell Separator
[0076] A polycarbonate container with outside dimensions
(length.times.width.times.thickness) of 41.times.41.times.18 mm
having a liquid outlet and a liquid inlet on the diagonal was
packed with 12 polyester nonwoven fabrics with an average fiber
diameter of 2.3 .mu.m on the inlet side and 25 polyester nonwoven
fabrics with an average fiber diameter of 12 .mu.m on the outlet
side. The packing density was 0.24 g/cm.sup.3, the effective
filtration area 9 2 cm.sup.2, and the effective filtration length
12.4 mm. In order to impart platelet permeability to the resulting
filter, coating with a hydrophilic polymer was carried out. In
detail, a 1% ethanolic solution of a hydroxyethyl methacrylate
dimethylaminoethyl methacrylate copolymer (molar ratio between
hydroxyethyl methacrylate and dimethylaminoethyl methacrylate=97:3)
was passed through the filter from the inlet side of the filter,
after which the filter was dried by introducing nitrogen gas
thereinto.
[0077] {circle over (2)} Preparation of a Recovering Liquid
[0078] A commercially available solution of dextran 40 in
physiological saline (Dextran 40 Injection-Midori, a trade name,
available from Green Cross Corp.) was incorporated with human serum
albumin to prepare a liquid containing 4% human serum albumin as
recovering liquid A. This recovering liquid A was diluted 1.2-fold
or 1.3-fold with physiological saline to obtain recovering liquid B
and recovering liquid C, respectively. The viscosities of the
recovering liquids are as follows: recovering liquid A 10.5
mPA.multidot.s, recovering liquid B 8.0 mPA.multidot.s, recovering
liquid C 5.3 mPA.multidot.s.
[0079] {circle over (3)} Cell Separation Procedure and Line
System
[0080] 200 Milliliters of cord blood collected from a placenta and
umbilical cord after delivery and containing 15 vol % CPD was
divided into four portions, and an experiment was carried out at 4
recovering liquid viscosity values (including that in Comparative
Example 1) by using the same blood divided.
[0081] As shown in FIG. 2, a blood bag was connected to the inlet
side of the cell separator 6 produced in the above item {circle
over (1)}, through a tube having between its ends a three-way
stopcock 9 having a bag for cell recovery 10 connected thereto, a
mesh chamber 8, and a diverging point to a tube equipped with a
spike 13 to be connected to a bottle of physiological saline for
rinsing. A drain bag 12 was connected to the outlet side of the
cell separator 6 through a tube having between its ends a three-way
stopcock 11 for connecting a syringe for recovery.
[0082] A fluid containing nucleated cells in the starting-blood bag
7 was introduced into the cell separator at a head of about 60 cm,
and a fluid containing erythrocytes and platelets which had flowed
out of the cell separator 6 was discharged into the drain bag 12.
Then, the spike 13 was stuck into the bottle of physiological
saline, and the clamp 14 was opened, whereby the inside of the
filter was rinsed with about 20 ml of physiological saline to rinse
away a slight amount of erythrocytes and platelets, which remained
in the filter. Subsequently, a 30-ml disposable syringe containing
25 ml of each recovering liquid was connected to the three-way
stopcock 11, and the three-way stopcock 11 was turned in such a
direction that the syringe communicated only with the cell
separator. The three-way stopcock 9 was turned in such a direction
that the cell separator 6 communicated only with the bag for cell
recovery 10. Then, the piston of the syringe was pushed to recover
cells captured in the cell separator, into the bag for cell
recovery 10.
[0083] {circle over (4)} Analysis
[0084] The number of nucleated cells, the number of mononuclear
cells, the number of erythrocytes, and the number of platelets were
determined with an automatic hemocytometer. The percentage of
CD34-positive cells based on the total number of nucleated cells
was measured by the use of FITC-labeled anti-CD34 antibody
according to a flow cytometry method comprising display on SSC-FITC
(Miyazaki et al. "Nichijo Shinryo to Ketsueki (Practical
Hematology)" Vol. 5, No. 2, pp. 21-24, 1995).
[0085] The recovery and the removal rate were calculated by the
following equations:
Recovery (%)=100.times.(number of recovered cells/number of cells
in starting cell population)
Removal rate (%)=100-100.times.(number of recovered cells/number of
cells in starting cell population)
[0086] {circle over (5)} Results
[0087] The time required for pushing the piston of the syringe
completely was 3 seconds. The linear speed was calculated to be
55.6 cm/min. The results are summarized in Table 1. It can be seen
that nucleated cells, mononuclear cells and CD34-positive cells
could be recovered at high percentages in the cell suspension
recovered, and that erythrocytes and platelets were removed at high
percentages.
1TABLE 1 Recovery (%) Recovering CD34- Removal rate (%) liquid
Nucleated Mononuclear positive Erythr- (mPa.s) cell cell cell ocyte
Platelet A (10.5) 75.2 90.2 97.0 99.0 88.0 B (8.0) 74.0 90.0 96.6
99.0 88.0 C (5.3) 73.0 89.6 95.5 99.0 88.0
[0088] The cells recovered by the use of the recovering liquid
could be subjected to cryopreservation according to the protocol
described in an instruction mannual for a cryopreservative agent
"CP-1" manufactured by Kyokuto Pharmaceutical Industrial Co., Ltd.
In detail, dimethyl sulfoxide was added to the recovered cell
suspension to adjust its final concentration to 5%, and the
resulting mixture was subjected to cryopreservation in a
deep-freezer at -80.degree. C. After 30 days of cryopreservation,
the mixture was rapidly thawed in a warm bath at 37.degree. C., and
the cell viability was measured by a conventional trypan blue
exclusion method and found to be maintained at a high value of
90.4%.
COMPARATIVE EXAMPLE 1
[0089] In this comparative example, results obtained by using a
recovering liquid with a low viscosity containing no dextran were
compared with those obtained in Example 1, though as in Example 1,
a cell-containing fluid was cord blood, cells to be recovered are a
mono-nuclear cell fraction containing hematopoietic stem cells, and
cells to be removed are erythrocytes and platelets.
[0090] {circle over (1)} Cell Separator
[0091] The same cell separator as in Example 1 was used.
[0092] {circle over (2)} Cell separation procedure and line
system
[0093] One of the portions of the cord blood obtained in Example 1
was used as starting cord blood. The process of Example 1 was
repeated except for using 25 ml of physiological saline as a
recovering liquid. The same line system as in Example 1 was used.
The viscosity of the recovering liquid was 1.0 mPa.multidot.s.
[0094] {circle over (3)} Analysis
[0095] The same analysis as in Example 1 was carried out.
[0096] {circle over (4)} Results
[0097] The time required for pushing the piston of the syringe
completely was 3 seconds. The results are summarized in Table 2.
The recoveries of nucleated cells, mononuclear cells and
CD34-positive cells in the cell suspension recovered were lower
than in Example 1.
2TABLE 2 Recovery (%) Recovering CD34- Removal rate (%) liquid
Nucleated Mononuclear positive Erythr- (mPa.s) cell cell cell ocyte
Platelet Physiological 31.0 40.0 45.0 99.0 89.7 saline (1.0)
EXAMPLE 2
[0098] This working example shows an example of cell separation in
the case where a cell-containing fluid was peripheral blood, cells
to be recovered are leukocytes, and cells to be removed are
erythrocytes and platelets.
[0099] {circle over (1)} Cell Separator
[0100] A polycarbonate container with outside dimensions
(length.times.width.times.thickness) of 41.times.41.times.18 mm
having a liquid outlet and a liquid inlet on the diagonal was
packed with 25 polyester nonwoven fabrics with an average fiber
diameter of 12 .mu.m on the inlet side and 12 polyester nonwoven
fabrics with an average fiber diameter of 2.3 .mu.m on the outlet
side. The packing density was 0.24 g/cm.sup.3, the effective
filtration area 9 cm.sup.2, and the effective filtration length
12.4 mm. In order to impart platelet permeability to the resulting
filter, coating with a hydrophilic polymer was carried out. A 1%
ethanolic solution of a hydroxyethyl
methacrylate.multidot.dimethyla- minoethyl methacrylate copolymer
(molar ratio between hydroxyethyl methacrylate and
dimethylaminoethyl methacrylate=97:3) was passed through the filter
from the inlet side of the filter, after which the filter was dried
by introducing nitrogen gas thereinto.
[0101] {circle over (2)} Cell Separation Procedure
[0102] Into the cell separator produced was introduced 50 ml of
whole peripheral blood (containing 15 vol % CPD) of a healthy
person through the liquid inlet by utilizing the head (about 60 cm;
flow rate about 5 ml/min.). Thereafter, 30 ml of physiological
saline was passed through the cell separator by means of head
(about 60 cm) in order to rinse away erythrocytes and platelets,
which remained in the cell separator. Then, 30 ml of a 3.5%
solution of a poly(vinylpyrrolidone) (average molecular weight:
360,000) in physiological saline was introduced into the cell
separator at a rate of 100 ml/min. through the liquid outlet by the
use of a pump, and cells were recovered through the liquid inlet.
The viscosity of this recovering liquid was 20.3
mPa.multidot.s.
[0103] {circle over (3)} Analysis
[0104] The number of leukocytes, the number of erythrocytes and the
number of platelets were determined with an automatic
hemocytometer.
[0105] {circle over (4)} Results
[0106] The results are summarized in Table 3. Leukocytes were
recovered at a high percentage in the cell suspension recovered,
and erythrocytes and platelets were removed at high percentages.
The linear speed was calculated to be 11.1 cm/min.
3TABLE 3 Recovery (%) Removal rate (%) Leucocyte Erythrocyte
Platelet 75.0 99.1 90.3
EXAMPLE 3
[0107] This working example shows an example of cell separation in
the case where a cell-containing fluid was cord blood, cells to be
recovered are hematopoietic stem cells (CD34-positive cells), and
cells to be removed are erythrocytes and platelets.
[0108] {circle over (1)} Cell Separator
[0109] A polycarbonate container with outside dimensions
(length.times.width.times.thickness) of 41.times.41.times.18 mm
having a liquid outlet and a liquid inlet on the diagonal was
packed with 12 polyester nonwoven fabrics with an average fiber
diameter of 12 .mu.m on the inlet side and 25 polystyrene nonwoven
fabrics with an average fiber diameter of 2.3 .mu.m having
anti-human CD34 monoclonal mouse antibody (clone name: Immu133,
available from Coulter Corp.; hereinafter abbreviated as "CD34
antibody") immobilized thereon, on the outlet side. The packing
density of the resulting filter was 0.2 g/cm.sup.3. The
immobilization of the anti-human CD34 monoclonal mouse antibody on
the polystyrene was carried out by the well-known haloacetamide
method proposed in JP-A-2-261833. In detail, polystyrene nonwoven
fabrics (previously cut to the above-mentioned dimensions) were
immersed in a treating solution prepared by adding 3.6 g of
hydroxy-methyliodoacetamide and 25 g of trifluoromethanesulfonic
acid to 165 ml of sulfolane, at room temperature for 5 hours to be
reacted, for the purpose of activating the polystyrene nonwoven
fabrics. The nonwoven fabrics thus activated were washed with
D-PBS, after which, in order to immobilize the antibody on them,
they were immersed for 2 hours in 10 ml of a CD34 antibody solution
having a concentration adjusted to 20 .mu.g/ml with D-PBS, and they
were washed with D-PBS and then freeze-dried, whereby the nonwoven
fabrics having the antibody immobilized thereon were obtained.
[0110] {circle over (2)} Preparation of a Recovering Liquid
[0111] A commercially available solution of dextran 40 in
physiological saline (Dextran 40 Injection-Midori, a trade name,
available from Green Cross Corp.) was incorporated with human serum
albumin to prepare a liquid containing 4% human serum albumin as a
recovering liquid. The viscosity of the recovering liquid was 9.8
mPA.multidot.s.
[0112] {circle over (3)} Cell Separation Procedure
[0113] A blood bag containing 50 ml of fresh human cord blood
(containing 15 vol % of an anticoagulant CPD) was connected to the
inlet side of the cell separator produced in the above item {circle
over (1)}, through a tube having between its ends, diverging points
to a physiological saline bag and a bag for cell recovery,
respectively. A blood bag for drain was connected to the outlet
side of the cell separator through a tube having a three-way
stopcock between the ends of the tube. Into the cell separator was
introduced 50 ml of the fresh cord blood by utilizing its fall
(about 60 cm), and an erythrocyte-containing fluid (also containing
CD34-negative cells and platelets) which had flowed out of the
filter was recovered into the drain bag. Then, 30 ml of
physiological saline was passed through the filter in order to
rinse away erythrocytes, platelets and CD34-negative cells, which
remained in the filter.
[0114] Subsequently, a syringe containing 30 ml of the recovering
liquid prepared in the above item {circle over (2)} was connected
to the three-way stopcock of the tube on the outlet side of the
cell separator, and the recovering liquid was introduced into the
cell separator by pushing the piston of the syringe, to recover the
captured cells into the bag connected to the inlet side.
[0115] {circle over (3)} Analysis
[0116] The same analysis as in Example 1 was carried out.
[0117] {circle over (4)} Results
[0118] The time required for pushing the piston of the syringe
completely was 3 seconds. The linear speed was 10 calculated to be
55.6 cm/min. The results are summarized in Table 4. It can be seen
that CD34-positive cells could be recovered at a high percentage in
the cell suspension recovered, and that erythrocytes, platelets and
CD34-negative cells were removed at high percentages.
4 TABLE 4 Recovery (%) Removal rate (%) CD34-positive CD34-negative
cell Erythrocyte Platelet cell 78 99.2 90.4 90
EXAMPLE 4
[0119] This working example shows an example of cell separation in
the case where a cell-containing fluid was cord blood, cells to be
recovered are a mononuclear cell fraction containing hematopoietic
stem cells, cells to be removed are erythrocytes and platelets, and
DNA for HLA typing was collected at the same time.
[0120] {circle over (1)} Cell Separator
[0121] The same cell separator as in Example 1 was used.
[0122] {circle over (2)} Preparation of Recovering Liquids
[0123] A commercially available solution of dextran 40 in
physiological saline (Dextran 40 Injection-Midori, a trade name,
available from Green Cross Corp.) was incorporated with human serum
albumin to prepare a liquid containing 4% human serum albumin as a
first recovering liquid (for cell recovery). Distilled water for
injection, and a hypotonic liquid was used as an additional
recovering liquid (for recovering cell constituents). The viscosity
of the first recovering liquid was 10.5 mPA.multidot.s.
[0124] {circle over (3)} Line System
[0125] The cell separation system shown in FIG. 3 was obtained by
incorporating the cell separator described in the above item
{circle over (1)} into lines. In this system, the connection of a
cell-containing fluid bag to the main body of the system of the
present invention, and the connection of a bag for recovering a
fluid which flows out through the outlet of the cell-capturing
means, to the main body of the system of the present invention were
made with spikes. A line for recovering cells from the inlet side
of the cell-capturing means was equipped with a freeze bag for
recovering cells for cell transfer, and a tube with a spike at the
end for recovering DNA for HLA typing into a conical tube. In this
line, the flow paths are changed by means of clamps.
[0126] {circle over (4)} Cell Separation Procedure
[0127] A cell separation procedure was carried out by using the
line system shown in FIG. 3.
[0128] Initially, clamp 21 was shut, a three-way stopcock 25 was
shut only in the direction of syringe connection, and clamps 27 and
28 were shut.
[0129] A spike 20 was stuck into a blood bag containing 50 ml of
fresh human cord blood (containing 15 vol % of an anticoagulant
CPD), and a spike 23 was stuck into an empty bag. When the clamp 21
was opened, the cell-containing fluid was supplied to the
cell-capturing means through the tube 22 of a line 16, and a
mononuclear cell fraction containing hematopoietic stem cells was
captured, and erythrocytes and platelets were discharged into the
empty bag through the tube 24 of a line 17.
[0130] After completion of the treatment of said cell-containing
fluid, the clamp 21 was shut and the spike 20 was pulled out, and
then stuck into a commercially available 100-ml bottle of
physiological saline. When the clamp 21 was opened, the
physiological saline rinsed away a slight amount of erythrocytes
and platelets, which remained in the cell-capturing means 15, and
the physiological saline was discharged through the line 17. Then,
the clamp 21 was shut. Next, a 30-ml syringe containing 25 ml of
the recovering liquid prepared in the above item {circle over (2)}
was connected to the three-way stopcock 25, after which the
three-way stopcock 25 was turned in such a direction that the
syringe communicated only with the cell-capturing means 15 through
a line 18, and the clamp 27 was opened. The piston of the syringe
was pushed to recover cells into a freeze bag 29 through a line 19.
Subsequently, the syringe was detached from the three-way stopcock
25, and another syringe containing 25 ml of distilled water for
injection was connected to the three-way stopcock 25. The clamp 27
was shut, and clamp 28 was opened, being attached to a tube 31
capable of communicating with the tube 32 of the line 19 through a
Y-tube 26. A conical tube was placed under a spike 30, after which
the distilled water for injection was introduced into the
cell-capturing means by pushing the piston of the syringe, to
disrupt the captured cells, and crude DNA in these cells was
recovered in the conical tube. The crude DNA recovered was purified
by a conventional method comprising deproteination using proteinase
K and phenol.multidot.chlorofo- rm method. Ps {circle over (5)}
Analysis
[0131] The numbers of cells were determined by the same method as
described in Example 1. The amount of the purified DNA was
determined by a conventional method comprising measuring absorbance
at 260 nm by means of a spectrophotometer (Nakayama et al., Cell
Technology, extra issue "Bio-experiment Illustrated" {circle over
(1)} Fundamentals of Molecular Biological Experiment, 1995).
[0132] {circle over (6)} Results
[0133] The time required for pushing the piston of the syringe
completely was 3 seconds. The linear speed was calculated to be
55.6 cm/min. The results are summarized in Table 5. It can be seen
that eukaryotic cells, mononuclear cells and CD34-positive cells
could be recovered at high percentages in the cell suspension
recovered, and that erythrocytes and platelets were removed at high
percentages. It can also be seen that the amount of the DNA
obtained was about 10 .mu.g, an amount sufficient for HLA
typing.
5TABLE 5 Recovery (%) CD34- Removal rate (%) Amount of Nucleated
Mononuclear positive Erythro- purified cell cell cell cyte Platelet
DNA (.mu.g) 75.0 90.4 97.2 98.9 88.3 9.8
EXAMPLE 5
[0134] This working example shows an example of cell separation in
the case where a cell-containing fluid was bone marrow, cells to be
recovered are a mononuclear cell fraction containing hematopoietic
stem cells, and cells to be removed are erythrocytes and
platelets.
[0135] {circle over (1)} Cell Separator
[0136] The same cell separator as in Example 1 was used.
[0137] {circle over (2)} Preparation of a Recovering Liquid
[0138] A commercially available solution of dextran 40 in
physiological saline (Dextran 40 Injection-Midori, a trade name,
available from Green Cross Corp.) was incorporated with human serum
albumin to prepare a liquid containing 4% human serum albumin as a
recovering liquid. The viscosity of the recovering liquid was 10.1
mPA.multidot.s.
[0139] {circle over (3)} Cell Separation Procedure and Line
System
[0140] As shown in FIG. 2, a blood bag containing 30 ml of bone
marrow (containing 15 units/ml of an anticoagulant heparin) was
connected to the inlet side of the cell separator 6 described in
the item {circle over (2)}, through a tube having between its ends
a three-way stopcock 9 having a bag for cell recovery 10 connected
thereto, a mesh chamber 8, and a diverging point to a tube with a
spike 13 to be connected to a bottle of physiological saline for
rinsing. A drain bag 12 was connected to the outlet side of the
cell separator 6 through a tube having between its ends a three-way
stopcock 11 for connecting a syringe for recovery. The fluid
containing nucleated cells in the starting-blood bag 7 was
introduced into the cell separator at a fall of about 60 cm, and a
fluid containing erythrocytes which had flowed out of the cell
separator 6 was discharged into the drain bag 12. Then, the spike
13 was stuck into the bottle of physiological saline, and the clamp
14 was opened, whereby the inside of the filter was rinsed with
about 20 ml of physiological saline to rinse away a slight amount
of erythrocytes and platelets, which remained in the filter.
Subsequently, a 30-ml disposable syringe containing 25 ml of the
recovering liquid was connected to the three-way stopcock 11, and
the three-way stopcock 11 was turned in such a direction that the
syringe communicated only with the cell separator. The three-way
stopcock 9 was turned in such a direction that the cell separator 6
communicated only with the bag for cell recovery 10. Then, the
piston of the syringe was pushed to recover cells captured in the
cell separator, into the bag for cell recovery 10.
[0141] {circle over (4)} Analysis
[0142] The number of nucleated cells, the number of mononuclear
cells, the number of erythrocytes and the number of platelets were
determined with an automatic hemocytometer. The percentage of
CD34-positive cells based on the total number of nucleated cells
was measured by the use of FITC-labeled anti-CD34 antibody
according to a flow cytometry method comprising development on
SSC-FITC (Miyazaki et al. "Nichijo Shinryo to Ketsueki (Routine
Diagnosis and Treatment, and Blood)" Vol. 5, No. 2, pp. 21-24,
1995).
[0143] The recovery and the removal rate are calculated by the
following equations:
Recovery (%)=100.times.(number of recovered cells/number of cells
in starting cell population)
Removal rate (%)=100-100.times.(number of recovered cells/number of
cells in starting cell population)
[0144] {circle over (5)} Results
[0145] The time required for pushing the piston of the syringe
completely was 3 seconds. The linear speed was calculated to be
55.6 cm/min. The results are summarized in Table 6. It can be seen
that nucleated cells, mononuclear cells and CD34-positive cells
could be recovered at high percentages in the cell suspension
recovered, and that erythrocytes and platelets were removed at high
percentages.
6TABLE 6 Recovery (%) Nucleated Mononuclear CD34-positive Removal
rate (%) cell cell cell Erythrocyte Platelet 74.3 91.2 97.6 99.0
88.0
[0146] Industrial Applicability
[0147] As described above, according to the present invention,
useful cells such as hematopoietic stem cells can be recovered with
high recovery by a simple and rapid procedure, and the thus
obtained cell-containing fluid can be subjected to cryopreservation
without a subsequent troublesome cell suspension preparation
procedure, and hence can be utilized in a laborsaving cell
processing in medical-care-related industries such as the field of
hematopoietic stem cell transplatation, the field of
adoptive-immuno therapy, etc.
* * * * *