JP3722523B2 - Erythrocyte removal system - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a system for removing red blood cells from a cell population containing red blood cells and hematopoietic stem cells and / or hematopoietic progenitor cells, such as bone marrow or peripheral blood.
[0002]
[Prior art]
Currently, bone marrow transplantation or peripheral blood stem cell transplantation therapy using peripheral blood is performed for hematopoietic disorders, which are the main side effects in chemotherapy of hematopoietic tumors such as leukemia and solid cancer. As is well known, when allogeneic transplantation is performed in these transplantation therapies, matching the HLA (leukocyte blood group) between the donor and recipient (patient) is most important, so the difference in ABO blood group (red blood cell group) Resolves by removing red blood cells from donor bone marrow or peripheral blood. Conventionally, a method of separating a leukocyte fraction (more specifically, a mononuclear cell fraction) containing hematopoietic stem cells and / or hematopoietic progenitor cells required for transplantation and a red blood cell fraction by a centrifuge as a means for removing red blood cells Has been done. In order to separate erythrocytes and mononuclear cells effectively, a centrifugal method using a specific gravity solution called density gradient centrifugation (for example, Ficoll manufactured by Pharmacia) is performed. Skill is required and complicated, such as the liquid level must never be disturbed. In addition, the use of a specific gravity liquid is expensive. Many attempts have been made to solve the complicated operation in Japanese Patent Laid-Open Nos. 61-84557 and 2-134564, but it is still simple, low cost, and removes red blood cells from bone marrow in a short time. There is no technology to do.
[0003]
[Problems to be solved by the invention]
An object of the present invention is to provide an erythrocyte removal system that can be operated easily and at low cost in a short time without using a centrifuge or a special reagent and that can be aseptically operated.
[0004]
[Means for Solving the Problems]
FIG. 1 is a schematic view of the red blood cell removal system of the present invention.
According to the present invention, the first means for storing the collected raw cell fluid, the filter means 2 for passing the red blood cells and capturing the white blood cells, and the means 3 for storing the red blood cell-containing liquid flowing out from the filter means 2 are respectively first. And a recovery liquid reservoir 4 connected to the third tube 8 branched from the second tube 7 downstream of the filter means, and a first upstream of the filter means. The leukocyte collection unit 5 connected to the fourth tube 9 branched from the tube 6 is provided, and flows only from the collected solution storage unit 4 to the filter means 2 at the branch point of the second and third tubes. And a means for causing the blood to flow only in the direction from the filter means 2 to the leukocyte collection liquid section 5 at the branching point of the first and fourth tubes. On blood cell removal system. Furthermore, the present invention relates to an erythrocyte removal system having means for rinsing the filter means before collecting leukocytes.
Examples of filter means used in the present invention include nylon wool, polyester non-woven fabric, hydroxyapatite beads, sponge polymer compounds, etc., but are not limited thereto, and it is a material that can collect leukocytes after capturing leukocytes. I just need it. However, if it is desired to remove platelets together with erythrocytes in order to increase the separation efficiency in the field of cell separation for the purpose of removing cells that cause complications in allogeneic bone marrow transplantation or cancer cells in autologous bone marrow transplantation, The surface charge is adjusted, for example, by a coating treatment that allows platelets to pass through the filter means of the invention. Means for flowing in one direction at the tube branch points of the second and third and first and fourth of the present invention include three-way stopcocks, dual check valves, clamps, etc., but three-way stopcocks are used. Is desirable.
Further, the recovery solution storage unit 4 contains a recovery solution for recovering leukocytes captured by the filter means. The recovered solution is a solution in which a protein such as human serum albumin or an anticoagulant is added to a buffer solution such as physiological saline, HBSS (Hanks solution), D-PBS (Dulbecco phosphate buffer solution) as necessary. Used.
Moreover, the raw material cell liquid used in the present invention is a liquid containing erythrocytes, hematopoietic stem cells and / or hematopoietic progenitor cells, and includes bone marrow, peripheral blood, umbilical cord blood, or those obtained by roughly separating these using a centrifuge. In the future, with the development of a technique for culturing and transplanting a small amount of hematopoietic stem cells using the stem cell amplification method, the presence or absence of erythrocyte removal is expected to increase further.
[0005]
[Example 1]
An example of an embodiment of the present invention will be described with reference to the drawings. Reference numeral 1 denotes a container (usually a blood bag) containing raw material cells, which is connected to the first tube 6. Reference numeral 4 denotes a bag containing the collected liquid, from which the collected liquid is supplied to the leukocyte removal filter 2. The third tube 8 is a tube that supplies the recovered liquid to the filter means 2 and is connected to the second tube 7. A clamp 14 is provided in the middle of the third tube, and a three-way stopcock 12 is provided at a branch point between the third tube 8 and the second tube 7. The tube 10 is a tube for introducing the rinse liquid into the filter means 2, and is branched from the first tube 6 by a Y-shaped tube or the like. There is a clamp 13 in the middle of the tube 10. It is desirable that the first tube 6 has a clamp 16 and a mesh trap 15. The fourth tube 9 is for introducing the collected cell fluid into the leukocyte collection unit 5. There is a three-way stopcock 11 at the branch point of the first tube 6 and the fourth tube 9. The filter means 2 is a filter that captures leukocytes while passing red blood cells (and platelets if platelets are to be removed). The bag used for each means 1, 3, 4, 5 can be connected to the tube with a spike or aseptic connector.
Next, the usage method will be described. First, bone marrow, peripheral blood buffy coat, umbilical cord blood and the like collected by a conventional method are put in the blood bag 1 in advance. At this time, the clamp 13 is in the “closed” state, the three-way stopcock 11 is in the “closed” direction of the tube 9, and the three-way stopcock 12 is in the “closed” state of the tube 8. The cell fluid in the blood bag 1 is introduced into the leukocyte removal filter 2 through the first tube 6. There, the white blood cells are captured, and the red blood cells and platelets pass through and are collected from the second tube 7 into the drainage bag 3. In addition, in order to wash away some red blood cells remaining in the filter 2, the clamp 13 is opened, and the collection liquid is collected from the collection bag 4 to the tube 10, the first tube 6, the leukocyte removal filter 2, and the second tube in this order. To the drainage bag 3. Next, the clamp 13 is “closed”, the direction of the second tube 7 (drainage bag side) of the three-way cock 12 is “closed”, and the direction of the first tube 6 (blood bag side) of the three-way cock 11 is Try to be “closed”. Then, the collected liquid bag 4 is crushed strongly to introduce the collected liquid into the leukocyte removal filter 2, the leukocytes captured by the filter are pushed out, and flow into the leukocyte collection bag 5 through the fourth tube 9.
The cell solution collected in the leukocyte collection bag 5 is left as it is or reduced in volume by concentrating operation, and then infused to the patient, or frozen and stored after adding necessary work such as addition of frost damage protective agent, and thawed after mass therapy. It is used as infusion to patients.
[0006]
【The invention's effect】
According to the present invention, it is possible to provide an erythrocyte removal system that does not use a centrifuge or a special reagent, can be operated in a simple manner at low cost in a short time, and can be aseptically operated.
[Brief description of the drawings]
FIG. 1 is a schematic view of a “red blood cell removal system” of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Blood bag 2 Leukocyte removal filter 3 Drainage bag 4 Collected solution bag 5 Leukocyte collection bag 6 1st tube 7 2nd tube 8 3rd tube 9 4th tube 10 Filter rinse tube 11, 12 Three-way stopcock 13 Clamp 14 Clamp 15 Mesh trap 16 Clamp

Claims (2)

Means 1 for storing the collected raw cell fluid, filter means 2 for passing red blood cells and capturing white blood cells, and means 3 for storing the red blood cell-containing liquid flowing out from the filter means 2 are respectively first and second. The recovery liquid storage unit 4 is connected in series via a tube and connected to a third tube 8 branched from the second tube 7 downstream of the filter means, and from the first tube 6 upstream of the filter means. Means for providing a leukocyte collection part 5 connected to the branched fourth tube 9 and allowing the second and third tubes to circulate only from the collected liquid storage part 4 to the filter means 2 at the branch point. At the branching point of the first and fourth tubes, there is provided a means for allowing the filter means 2 to flow only in the direction of the leukocyte collection section 5. Temu. The erythrocyte removal system according to claim 1, further comprising a rinsing tube (10) for the filter means (2) connected from the collected liquid storage part (4) to the first tube (6).

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