JPH09253195A - Device for separating stem cell and precursor cell from umbilical cord blood - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device to separate stem cells and precursor cells from umbilical cord blood in which mingling of bacteria is eliminated, and in which loss of large quantity of cells by separating operation can be prevented. SOLUTION: A device comprises a parent bag 3 containing umbilical cord blood to be separated to blood components by centrifugal separation, a first child bag 5 to which blood plasma in the parent bag 3 is moved to be contained, a means to separate the blood plasma in the first child bag 5 to a blood plasma component and a leucocyte component, a second child bag 9 to which the blood plasma component at an upper layer of the first child bag 5 is moved to be contained, a freezing bag 11 to which the leucocyte component, in the first child bag 5 is moved to be frozen and stored, and tubes making these bags communicate with each other for moving the separated blood components, where stem cells and precursor cells are separated from the umbilical cord blood.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apparatus for collecting umbilical cord blood from a human fetus or newborn umbilical cord and placenta, and separating stem cells and progenitor cells from the cord blood.

[0002]

Umbilical cord blood collected from human fetal or neonatal umbilical cord and placenta contains a large amount of hematopoietic stem cells and progenitor cells. Therefore, gene therapy, bone marrow transplantation treatment,
Autologous blood transfusions, cultured blood products, etc. have been found to have significant therapeutic effects. Umbilical cord blood, unlike bone marrow fluid, is easier to collect, less likely to be infected with bacteria or viruses, and transplanted cells attack the patient's tissues compared to adult immune cells in the bone marrow fluid. Host disease (GVH
It has the advantage that complications such as D) are unlikely to occur.

[0003] Conventional umbilical cord blood is collected by cutting the umbilical cord and collecting it by using uterine contraction, or by introducing a needle or an insertion tube into the umbilical cord blood vessel or placenta blood vessel and spontaneously dropping by gravity using the head. , Was collected into the container by the suction force of a syringe or the like. A blood preservation solution is infused to preserve the blood in good condition.
Since the amount of blood collected differs depending on the placenta, the amount of blood preservation solution must be calculated for each amount of collected blood, and the quantified blood preservation solution must be injected into the blood collection container, which is very complicated in operation. . In addition, a large amount of cells are lost in the process of separating the collected umbilical cord blood into blood components, so a cryoprotectant solution such as dimethylsulfoxide is added to the total amount of cord blood and cryopreserved in liquid nitrogen and thawed during transplantation. However, since an approximately equal amount of antifreezing agent solution is added to the total amount of cord blood, the volume increases and the antifreezing agent, which is harmful to the recipient, is also injected. It was

[0004]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and freezes leukocyte components obtained by separating cord blood collected from umbilical cords and placenta of human fetuses or newborns into blood components. By saving
It is an object of the present invention to provide an apparatus for separating stem cells and progenitor cells from cord blood, which prevents the loss of a large amount of cells due to the separation operation without the risk of bacterial contamination.

[0005]

[Means for Solving the Problems] That is, the present invention provides a parent bag for containing cord blood and separating it into blood components by centrifugation, a first child bag for transferring and storing plasma of the parent bag, Means for separating plasma of the first child bag into plasma component and white blood cell component, second child bag for transferring and containing the plasma component of the upper layer of the first child bag, and transfer of white blood cell component of the first child bag It is an apparatus for separating stem cells and progenitor cells from umbilical cord blood, which comprises a frozen bag for cryopreservation and a tube for transferring the separated blood components in communication with each bag.

Further, in the apparatus for separating stem cells and progenitor cells from umbilical cord blood, the present invention separates stem cells and progenitor cells from umbilical cord blood obtained by injecting a cryoprotectant into a first child bag containing only white blood cell components. It is a device that does.
Furthermore, the present invention relates to a device for separating stem cells and progenitor cells from umbilical cord blood, comprising: means for thawing and liquefying a white blood cell component cryopreserved in a freezing bag into a liquid phase; Is a device for separating stem cells and progenitor cells from umbilical cord blood.

Umbilical cord blood is collected from the umbilical cord and placenta of a human fetus or newborn and injected into a parent bag containing an anticoagulant in advance to prevent coagulation, and then the erythrocyte precipitation reagent is added to the parent bag and then centrifuged. Separation separates cord blood into plasma and red blood cell components. Then, the upper layer plasma is transferred to the first child bag, and the plasma is centrifuged to separate it into a plasma component and a white blood cell component. After transferring the plasma component of the upper layer to the second child bag, the antifreezing agent is added to the white blood cell component remaining in the first child bag. Then, the white blood cell component containing the antifreezing agent is transferred to a freezing bag and frozen in liquid nitrogen for storage. Next, the frozen white blood cell component containing the antifreezing agent is thawed into a liquid phase, the antifreezing agent is washed to dilute the contents, and the precipitated stem cells and progenitor cells are separated by centrifugation to obtain them.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is an explanatory view showing an embodiment of the present invention. In the figure, 1 is a blood collecting needle, 2, 4, 6, 8 and 10 are tubes, 3 is a parent bag, 5 is a first child bag, 7 is a switching valve, 9 is a second child bag, and 11 is a frozen bag. , 12 is a roller clamp, and 13 and 14 are clamps.

The device of the present invention will be described with reference to FIG. 1. Umbilical cords and placenta of human fetuses or newborns are collected with an injection needle 1 to collect cord blood, and a parent bag 3 containing an anticoagulant such as CPD solution. Is injected from tube 2. After injecting cord blood into the parent bag 3, the tube 2 is sealed with the clamp 13. At this time, if necessary, hydroxyethyl starch, dextran, gelatin, polyethylene glycol-
A red blood cell aggregating agent such as polyvinyl alcohol or polyvinylpyrrolidone may be added to the parent bag 3. Then, the cord blood in the parent bag 3 is centrifuged with a centrifuge to separate it into plasma and blood cell components. Plasma, which is the supernatant of the parent bag 3, is transferred to the first child bag 5 through the tube 4 while controlling the flow rate with the tube clamp 12. When the injection of plasma into the first child bag 5 is completed, the connecting tube 4 between the parent bag 3 and the first child bag 5 is melted and the parent bag 3 is cut off.

Next, the first child bag 5 containing the plasma component
Were centrifuged with a centrifuge, the sedimented white blood cell component was left in the first child bag 5, and the upper plasma component was placed in the tube 6 and the switching valve 7.
Then, it is transferred to the second child bag 9 via the tube 8.
Examples of the switching valve 7 include a three-way stopcock and a Y-shaped tube. Parent bag 3, first child bag 5 and second child bag 9
Examples of materials include polyvinyl chloride, polyurethane, polyethylene, ethylene-vinyl acetate copolymer, ethylene
Examples include α-olefin copolymers, polyfluorocarbons and polyimides. Subsequently, the antifreezing agent is slowly added to the white blood cell components remaining in the first child bag 5 over time. Examples of the antifreezing agent include dimethyl sulfoxide, which is preferably diluted with dextran at 50%.

Next, the white blood cell component containing the antifreezing agent stored in the first child bag 5 is stored in the freezing bag 11 via the tube 8, the switching valve 7 and the tube 10. Examples of the freezing bag 11 include polyethylene, ethylene-vinyl acetate copolymer, fluororesin, and polyimide, which have good low temperature resistance. When the white blood cell component containing the antifreezing agent is completely injected into the freezing bag 11, the tube 10 is sealed with the clamp 14.
The tube 10 is melted and the frozen bag 11 is separated from other bags. The freezing bag 11 comprises a first chamber 15 having a large volume and a second chamber 16 having a small volume. The two chambers are divided by an upper portion 17 and a lower portion 18 being heat-sealed. The white blood cell component of the second chamber 16 having a small volume is used for confirming or testing whether or not the recipient is HLA-compatible. A mouth portion is provided in each of the first chamber 15 and the second chamber 16. Further, the gap between the first chamber 15 and the second chamber 16 becomes a cut line, and the first chamber 15
And the second chamber 16 can be separated.

The white blood cell component contained in the freezing bag together with the antifreezing agent is gradually cooled to an extremely low temperature in liquid nitrogen and stored for a long period of time. Then one particular frozen bag 11
Once it is decided to use the stem cells and progenitor cells, the frozen bag 11 containing the stem cells is immersed in, for example, a 37 ° C. water bath, and the leukocyte and stem cell suspension are melted and phase-converted from the solid phase to the liquid phase. . Thereafter, the cryoprotectant added before freezing the leukocyte components is removed by washing. The washing of the antifreezing agent is preferably performed using an isotonic liquid, and after dilution, the mixture is centrifuged to remove the upper layer to separate the precipitated stem cells and progenitor cells. After thawing the leukocyte and stem cell suspension, inject such leukocyte concentrate in v
Culturing on itro can increase the number of active stem and progenitor cells.

[0013]

EFFECTS OF THE INVENTION According to the present invention, the white blood cell component obtained by the separation of cord blood by the closed system is frozen and stored, so that there is no fear of contamination of bacteria and the loss of a large amount of cells due to the separation operation is prevented. Stem cells and progenitor cells can be obtained. Furthermore, the apparatus of the present invention can supply the recipient with stem cells and progenitor cells from which harmful substances such as anti-freezing agents and released hemoglobin that cause adverse effects on the recipient are removed.

[Brief description of drawings]

FIG. 1 is an explanatory view showing one embodiment of the present invention.

[Explanation of symbols]

 1 Blood collection needle 3 Parent bag 5 1st child bag 7 Switching valve 9 2nd child bag 11 Frozen bag

Claims (3)

[Claims] 1. A parent bag for containing cord blood and separating it into blood components by centrifugation, a first child bag for transferring and storing the plasma of the parent bag, and a plasma component of the plasma of the first child bag. And a means for separating into white blood cell components, a second child bag for transferring and containing the plasma component in the upper layer of the first child bag, and a frozen bag for transferring and storing the white blood cell components in the first child bag in a frozen state. A device for separating stem cells and progenitor cells from umbilical cord blood, which comprises a tube for communicating separated bags and transferring a separated blood component. 2. The device for separating stem cells and progenitor cells from umbilical cord blood according to claim 1, wherein a cryoprotectant is injected into a first child bag containing only leukocyte components. 3. A method comprising thawing a white blood cell component frozen and stored in a freezing bag into a liquid phase and washing the antifreezing agent to obtain stem cells and progenitor cells.
The device for separating stem cells and progenitor cells from umbilical cord blood according to item 2.