JP4061715B2 - Leukocyte separation and concentration method - Google Patents

Description

【００１６】
【発明の効果】
以上説明してきたことから明らかなように、本発明の方法を採用することにより、血液母液から白血球製剤を安全かつ無菌的に効率よく分離し濃縮して、造血幹細胞製剤として凍結保存することができる。また、製剤に含まれる造血幹細胞数を精度良く管理することができ、さらに製剤中への赤血球の混入を極力防止することができる。
【図面の簡単な説明】
【図１】本発明で使用する血液母液処理バッグの一例を示す説明図である。
【図２】本発明で使用する血液母液処理バッグの他の例を示す説明図である。
【符号の説明】
１ 親バッグ
２ 採血チューブ
２１ 採血針
３、４ 子バッグ
５ 凍結バッグ
６１、６３、６４、６５ 連結チューブ
７３、７４、７５ クランプ
８ ポート[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for separating and concentrating leukocytes from a blood mother liquor containing blood components, and in particular, leukocytes containing cord blood, bone marrow fluid, hematopoietic stem cells and / or hematopoietic progenitor cells can be safely and aseptically obtained from peripheral blood or the like. In particular, it relates to a method for efficiently, rapidly and economically collecting, separating and concentrating blood.
[0002]
[Prior art]
In recent years, hematopoietic stem cell transplantation by umbilical cord blood transplantation, bone marrow transplantation or peripheral blood stem cell transplantation is the cure for blood diseases such as aplastic anemia and leukemia, solid cancers such as breast cancer and testicular cancer, or autoimmune diseases such as rheumatism. It is getting attention as a therapy.
In performing hematopoietic stem cell transplantation, leukocytes containing hematopoietic stem cells and hematopoietic progenitor cells are collected safely and aseptically, efficiently, rapidly and economically from a blood mother liquor such as cord blood, bone marrow or peripheral blood. Need to be separated, concentrated and prepared for transplantation.
In the blood mother liquor treatment process, in order to accurately grasp the number of hematopoietic stem cells to be transplanted, it is necessary to carry out a treatment operation while grasping the volume of blood components transferred between bags, It is necessary to eliminate the cumbersome work as much as possible to improve the work efficiency, and to prevent the contamination and cost increase of the preparation due to the troublesome work.
[0003]
As a method of collecting leukocytes, separating, concentrating and cryopreserving them, conventionally, blood was collected using a syringe under strict care not to contaminate the blood mother liquor, and transferred to a clean test tube. After adding the erythrocyte sedimentation agent and performing a light centrifugation, transfer only the supernatant to a clean test tube, add a freezing protection agent and store it frozen (test tube method), or collect the blood mother solution in a blood collection bag After adding the erythrocyte sedimentation agent and performing light centrifugation, connect the plastic needle of the connection tube of the leukocyte bag to the port of the blood collection bag, and transfer the concentrated leukocyte layer in the blood collection bag to the leukocyte bag. Note that the leukocyte layer is concentrated to an appropriate liquid volume by centrifuging and removing the supernatant, and subjected to treatment such as prevention of freezing damage and the like, and then cryopreserved (Bag method, WO 96/17514) ) Etc. are known That.
[0004]
In general, when producing a biologic such as a hematopoietic stem cell preparation, it is extremely difficult to sterilize the biologic, and therefore it is necessary to make all the manufacturing processes a closed system in order to prevent contamination by bacteria.
However, in the test tube method, when blood mother liquor is transferred from a syringe to a test tube in the test tube method, when the test tube is sealed and frozen and stored, and in the bag method, blood or the like is removed from the blood collection bag. When transferring to a bag, it includes a process in which blood directly touches the outside air, which may be contaminated by bacteria present in the environment, and it is never satisfactory as a blood mother liquor treatment method. .
Therefore, in order to produce a hematopoietic stem cell preparation free from contamination, it is desired to develop a new method for treating blood mother liquor on the premise of complete aseptic operation.
[0005]
Further, at the time of umbilical cord blood transplantation, it is required to eliminate or minimize the mixing of erythrocytes, which may cause side effects. Therefore, it is conceivable to apply a method of extracting red blood cells from a connecting tube opened to the bottom after centrifuging the stored parent bag in the blood mother liquor (so-called top and bottom method, see Japanese Patent Publication No. 63-20144). In this method, since red blood cells can be removed without disturbing the interface between the white blood cell layer and the red blood cell layer, the mixing of red blood cells can be reduced. In addition, aseptic operation can also accurately grasp the number of transplanted hematopoietic stem cells.
However, when the top-and-bottom method was simply applied to the blood mother liquor, the reason was not clear, but satisfactory results were not always obtained with respect to red blood cell contamination.
[0006]
[Problems to be solved by the invention]
The present invention has been made in view of the above circumstances, and can prevent contamination and contamination of erythrocytes as much as possible, and can accurately ascertain the number of hematopoietic stem cells to be transplanted. An object is to provide a separation and concentration method.
[0007]
[Means for Solving the Problems]
As a result of intensive studies, the present inventor has found that the above-mentioned problems can be solved by further lightly centrifuging the leukocyte layer fractionated by light centrifugation and fractionating it into a plasma layer and a leukocyte concentrated liquid layer. Completed the invention. That is, the present invention is such that a freezing bag , a first child bag, and a second child bag are connected to a parent bag provided with a blood collection tube via a connecting tube, and the first bag is the parent bag. The following steps (a) to ( g ) are used, which are connected by a connecting tube attached to the upper part of the bag, and the parent bag uses a blood mother liquor treatment bag containing collected umbilical cord blood. A method for separating and concentrating leukocytes. And the parent bag was inverted so that the bottom of (a) parent bag is on top and light centrifugation or hanging standing, separating blood mother liquor in the upper leukocyte layer and a lower erythrocytes layer. ( B ) With the parent bag turned upside down, the lower red blood cells are transferred to the first child bag via a connecting tube leading to the first child bag. (C) separating only the first child bag and poured transfer of red blood cells from the parent bag blown the connection tube leading to the first child bag. ( D ) The parent bag is erected so that the bottom of the parent bag from which red blood cells have been removed is positioned downward, and lightly centrifuged to separate the leukocyte layer into an upper plasma layer and a lower leukocyte concentrated liquid layer. ( E ) Slowly transfer the upper plasma layer to the second child bag until the volume of the parent bag reaches a predetermined amount. ( F ) A predetermined amount of antifreezing agent is aseptically added to the parent bag containing the leukocyte concentrate. ( G ) The leukocyte concentrate containing the cryoprotectant is transferred to the freezing bag, the connecting tube connected to the freezing bag is melted, and the freezing bag is separated from the parent bag. Each process. A predetermined amount of erythrocyte sedimenting agent may be added to the parent bag from which the blood mother liquor has been collected before step ( a ).
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Next, embodiments of the present invention will be described with reference to the drawings. 1 and 2 are explanatory views showing an example of a blood mother liquor treatment bag used in the present invention. The blood mother liquor treatment bag shown in FIG. 1 includes a blood collection tube 2 provided with a blood collection needle 21 at the tip, a first child bag 4 , a second child bag 3 , a freezing bag, and a parent bag 1 containing a blood anticoagulant. 5, the blood collection tube 2, the first child bag 4 , the second child bag 3 , and the freezing bag 5 are all connected on the top surface. Here, 61, 63, 64 and 65 are connecting tubes, 73, 74 and 75 are clamps, and 8 is a port for adding an erythrocyte sedimentation agent. The blood mother liquor treatment bag shown in FIG. 2 is the one in which the second child bag 3 and the freezing bag 5 in FIG. 1 are connected at the bottom.
[0009]
The case where the blood mother liquor processing bag shown in FIG. 1 is used will be described. First, umbilical cord blood is collected through a blood collection tube 2 in a parent bag 1 (capacity 200 ml, containing CPD solution 28 ml as a blood coagulant). After collecting the blood mother liquid, if necessary, a predetermined amount of erythrocyte sedimentation agent (for example, DMSO 5 ml) may be added to the parent bag 1 through the port 8, and a test sample may be collected from the port 8. Next, the attachment portion of the connection tube 61 that connects the parent bag 1 to the first child bag 4 , the second child bag 3 , (capacity 150 ml) is turned down, that is, the bottom portion is up. Invert and lightly centrifuge (step a ). As a result, a red blood cell layer having a large specific gravity settles in the lower layer of the parent bag 1, a clear interface is formed between the white blood cell layer and the red blood cell layer, and the blood mother liquor is separated into the upper white blood cell layer and the lower white blood cell layer. When erythrocyte sedimentation agent is added, erythrocytes settle in a short time, but when sufficient time is allowed, even if erythrocyte sedimentation agent is not added, it can be suspended and left still without light centrifugation. However, red blood cells settle and blood mother liquor can be separated into white blood cell layers and red blood cell layers.
[0010]
Next, with the parent bag 1 turned upside down, the lower red blood cells are transferred to the first child bag 4 via the connecting tubes 61 and 64 using a head or a separation stand (not shown). (Step b ). At this time, it is necessary to infuse gently and slowly while taking care not to contaminate the leukocytes in the supernatant. In this transfer operation, since the attachment portion of the connection tube 61 leading to the first child bag 4 to the parent bag 1 is at the bottom, the lower red blood cells are transferred to the first child bag 4 using a head. When pouring, the interface between the white blood cell layer and the red blood cell layer descends without being disturbed while keeping the level. Accordingly, since the upper layer leukocytes are not mixed into the red blood cell layer, most of the lower layer red blood cells can be transferred to the first child bag 4. The first child bag 4 into which the lower red blood cells have been transferred is separated from the parent bag 1 by fusing the connecting tube 64 connected thereto, for example, using a tube sealer (not shown) or the like (step c ). ,Discard.
[0011]
Next, the parent bag 1 from which the red blood cells are removed and the leukocyte layer remains is erected with the attachment portion of the connecting tube 61 communicating with the first child bag 4 facing up, that is, with the bottom portion facing down. And lightly centrifuge (step d ). Thereby, the target white blood cells are further condensed and settled in the lower layer of the parent bag 1, and the supernatant becomes a plasma layer containing no white blood cells. Using a separation stand (not shown) for the plasma layer of the supernatant, while observing the weight of the parent bag 1 (usually observing the weight by placing the parent bag 1 on an electronic balance), Gently and slowly infuse the second child bag 3 until the volume of 1 reaches a predetermined amount (step e ). Here, the most important management item for the quality control of the hematopoietic stem cell preparation is the management of the number of white blood cells in the preparation, and therefore accurate white blood cell volume management is required. Therefore, according to the present invention, the blood mother liquor treatment bag is connected to the parent bag 1 and the second bag so that an appropriate amount of plasma once transferred to the second child bag 3 can be aseptically and accurately returned to the parent bag 1 . The child bag 3 is integrally connected by connecting tubes 61 and 63. The number of white blood cells in the parent bag 1 can be accurately calculated from the volume of the white blood cell concentrate adjusted to a predetermined volume and the test result of the separately sampled white blood cell concentrate. In step e , since leukocytes have settled on the bottom of the parent bag 1, the loss of leukocytes that occurs during transfer to the second child bag 3 can be extremely reduced.
[0012]
Next, a predetermined amount of antifreezing agent is aseptically added through the port 8 to the parent bag 1 containing the white blood cell concentrate (step g). For example, in the case of an antifreeze agent in which DMSO and dextran 40 are mixed at a ratio of 1: 1, the amount of the antifreeze agent added is 5 ml with respect to 20 ml of the leukocyte concentrate. The concentrated leukocyte solution that is concentrated to a predetermined volume and contains a blood coagulant and an antifreezing agent is transferred to the freezing bag 5 using a drop, and when the transfer is completed, the connecting tube 65 is melted to freeze the freezing bag. 5 is separated from the parent bag 1 (step h). The obtained hematopoietic stem cell preparation is stored frozen in liquid nitrogen according to a conventional method.
In the case of using the blood mother liquor treatment bag shown in FIG. 2, the same operation as that in the case of using the blood mother liquor treatment bag shown in FIG. That is, when the blood mother liquor processing bag shown in FIG. 2 is used, in step b, it is not necessary to invert the blood mother liquor processing bag, and light centrifugation is performed with the bottom upright.
[0013]
[Examples 1-2] Place the placenta and umbilical cord delivered after childbirth into a sterilized tray and transfer it to a clean umbilical cord blood collection room, and then place the umbilical cord side down using a placenta holder and placenta sheets. The blood mother liquor is hung on the stand and the surface of the umbilical cord is thoroughly disinfected, and then the blood mother liquor according to the method of the present invention is used using the blood mother liquor treatment bag as shown in FIG. 1 (Example 1) and FIG. 2 (Example 2). After separating and concentrating leukocytes, the freezing bag 5 containing leukocytes was cryopreserved in liquid nitrogen according to a conventional method. 6 units each of the thus obtained hematopoietic stem cell preparations were prepared, each thawed in warm water at 37 ° C., and tested for sterility and quality (nucleated cell recovery rate, hematopoietic progenitor cell recovery rate and color). However, the results shown in Table 1 were obtained. In this test, HES40 (6% hydroxyethyl starch, manufactured by Hishiyama Pharmaceutical Co., Ltd.) corresponding to 20% of the collected blood was collected from port 8 using a 30 ml syringe after blood mother liquor was collected in parent bag 1. It is added to the parent bag 1. Further, the centrifugation in the steps a and d was performed at a centrifugal force of 70 G for 5 minutes and at a centrifugal force of 400 G for 5 minutes, respectively. The sterility test was carried out using a blood test bottle (manufactured by Becton Dickinson) and cultured for 2 weeks under an aerobic atmosphere at 35 ° C. and an anaerobic condition at 25 ° C. The number of nucleated cells is measured using an automatic blood cell counter (manufactured by Coulter), and the number of hematopoietic progenitor cells is cultured for 2 weeks in an atmosphere of 37 ° C. and 5% CO ₂ concentration using methylcellulose medium. Observed counts were made below. The degree of red blood cell contamination was determined by visual comparison of the redness (darkness) of the preparation.
[0014]
[Comparative Examples 1-3]
Place the placenta and umbilical cord delivered after childbirth into a sterilized tray and transfer it to a clean umbilical cord blood collection room. After thoroughly disinfecting the surface, according to the conventional bag method (Comparative Example 1) and test tube method (Comparative Example 2), the bag method was applied to a blood mother liquor treatment bag as shown in FIG. 2 (Comparative Example 3). ) After separating and concentrating leukocytes from each blood mother liquor, the freezing bag 5 containing leukocytes was cryopreserved in liquid nitrogen according to a conventional method. Six units each of the hematopoietic stem cell preparation thus obtained were prepared, each was thawed in warm water at 37 ° C. and tested in the same manner as in Test Example 1, and the results shown in Table 1 were obtained.
From the table, it can be seen that the quality of the hematopoietic stem cells according to the present invention is superior in sterility, nucleated cell recovery rate, and hematopoietic progenitor cell recovery rate and less erythrocyte contamination compared to the conventional method.
[0015]
[Table 1]

[0016]
【The invention's effect】
As is clear from the above description, by adopting the method of the present invention, the leukocyte preparation can be safely and aseptically and efficiently separated and concentrated from the blood mother liquor and cryopreserved as a hematopoietic stem cell preparation. . In addition, the number of hematopoietic stem cells contained in the preparation can be controlled with high accuracy, and contamination of erythrocytes into the preparation can be prevented as much as possible.
[Brief description of the drawings]
FIG. 1 is an explanatory view showing an example of a blood mother liquor treatment bag used in the present invention.
FIG. 2 is an explanatory view showing another example of a blood mother liquor treatment bag used in the present invention.
[Explanation of symbols]
1 Parent bag 2 Blood collection tube 21 Blood collection needle 3, 4 Child bag 5 Freezing bag 61, 63, 64, 65 Connection tube 73, 74, 75 Clamp 8 port

Claims (1)

A freezing bag, a first child bag, and a second child bag are connected to the top surface of the parent bag equipped with a blood collection tube via a connection tube,
The first bag is connected by a connecting tube attached to the top of the parent bag,
A method for separating and concentrating leukocytes comprising the following steps (a) to (g), wherein a blood mother liquor treatment bag containing collected umbilical cord blood is used as the parent bag.
(A) The parent bag is turned upside down so that the bottom of the parent bag is up and left to stand by light centrifugation or hanging, and the umbilical cord blood mother liquor is separated into an upper leukocyte layer and a lower erythrocyte layer.
(B) The erythrocytes in the lower layer are transferred to the first child bag via a connecting tube that leads to the first child bag while the bottom of the parent bag is turned upside down.
(C) Only the first child bag into which the erythrocytes have been transferred is separated from the parent bag by fusing the connecting tube leading to the first child bag.
(D) The parent bag is erected so that the bottom of the parent bag from which red blood cells have been removed is down, and lightly centrifuged to separate the leukocyte layer into an upper plasma layer and a lower leukocyte concentrated liquid layer.
(E) Slowly transfer the upper plasma to the second child bag until the volume of the parent bag reaches a predetermined amount.
(F) A predetermined amount of antifreezing agent is aseptically added to the parent bag containing the leukocyte concentrate.
(G) The leukocyte concentrate containing the cryoprotectant is transferred to the freezing bag, the connecting tube connected to the freezing bag is melted, and the freezing bag is separated from the parent bag.

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