Method for separating cells

Details Method for separating cells Method for separating cells

1999-07-19

2001-07-31

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Maintaining blood or sperm in a physiologically active state...

C435S286500, C435S308100, C435S261000

Reexamination Certificate

active

06268119

ABSTRACT:

TECHNICAL FIELD
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
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.
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 necesary.
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.
Dextran is a polysaccharide composed of glucose units as monomer units mainly by &agr;-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.
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, one 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.
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.
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.
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
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·s and not less than 5 mPa·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.
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·s and not less than 5 mPa·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.
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 wi

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