JP4034830B2 - Cell separation material - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a material for separating platelets or leukocytes from blood, blood products or a liquid containing blood cells.
[0002]
[Prior art]
As a method for separating platelets or leukocytes from blood, blood products or a liquid containing blood cells, there are a centrifugal separation method and a filtration method using a filter. Centrifugation methods mainly used to separate and collect platelets from whole blood include a method of centrifuging blood in a blood bag and a method using a blood component separation device. However, there are disadvantages such as complicated and time-consuming operations for centrifugal separation and transfer to a separation bag.
[0003]
In addition, the blood component separation apparatus is fully automatic, and the operation of the blood sampler is simplified. However, there are drawbacks such as a high cost of the apparatus and a long donor restraint time. Furthermore, as a common drawback of the centrifugal separation method, blood cells are separated only by the difference in specific gravity, so the selectivity of blood cells is low, and blood cells other than the intended purpose are mixed.
[0004]
Many patents have been disclosed regarding filtration methods using filters mainly used for capturing and collecting leukocytes from blood products (Japanese Patent Publication Nos. 58-54125, 58-54130, 61-39060). No. 63, JP-B-63-26089, etc.), all methods are methods of washing blood cells after capturing leukocytes or lymphocytes with a filter medium, and this operation is performed without changing the interaction between the filter medium and blood cells. The recovery and function retention were low and not satisfactory.
[0005]
Furthermore, as a method of solving these disadvantages, blood cells are adsorbed on a polymer gel whose affinity with blood cells changes according to external stimuli, and the blood cells are lost by losing the affinity between blood cells and gel by external stimuli. Although there is an attempt to recover, the gel alone has the disadvantages that the strength is weak and the surface area that can be effectively used is small.
[0006]
[Problems to be solved by the invention]
The present invention has been made in order to solve the above-described problems, and does not require a complicated operation and a high-cost device, does not contain blood cells other than the intended purpose, has a low burden on the donor, and is high. An object of the present invention is to provide a cell separation material that is selective and ensures high recovery and function retention.
[0007]
[Means for Solving the Problems]
In view of the above points, the present inventors have conducted extensive research, and as a result, the polymer porous body or the fiber assembly in which the surface is coated with a polymer gel whose affinity for cells changes according to external stimuli. It has been found that the above problems can be solved by using as a cell separation material.
[0008]
That is, the present invention is a material used for a filter that selectively captures and collects platelets or leukocytes from blood or blood products, and uses a polymer porous body or fiber assembly having an average pore diameter of 1 to 60 μm as a base material, The surface of the base material is coated with a polymer gel having properties such as temperature sensitivity, light sensitivity, and electrical sensitivity, and the affinity with platelets or leukocytes is changed by swelling / contraction of these gels. Thus, the platelets or leukocytes are absorbed and desorbed.
[0009]
The porous polymer used for the substrate has continuous pores such as polyurethane, polyvinylidene fluoride, polysulfone or polyethersulfone, polyamide, polyester, polyether polyamide, ethylene vinyl alcohol, polyethylene, polypropylene, polyvinyl formal, polystyrene, etc. Any material can be used as long as it is a material capable of forming a porous body.
[0010]
When using a fiber assembly as the base material, organic synthetic fibers such as polyamide, polyester, polyacrylonitrile, polytetrafluoroethylene, semi-synthetic fibers such as acetate, recycled artificial fibers such as copper ammonia rayon, and inorganic such as glass fibers Natural fibers such as fiber, cotton, silk, and wool can be used.
[0011]
As the form of the substrate, there are membranes such as flat membranes and hollow fiber membranes made of porous bodies, or simply lumps of porous bodies, aggregates of fibers, fibers made of nonwoven fabrics, woven fabrics, knitted fabrics, etc. However, any form suitable for separating and collecting the target blood cells may be used, but a fiber column, a fiber filter, and a porous filter, which are generally used for leukocyte removal filters, are easy to use.
[0012]
The polymer gel coated on the surface of the substrate is obtained by the following polymer or copolymer. Polymers that can be used are not limited to these compounds, but in the case of temperature sensitivity, for example, acrylamide or (meth) acrylamide compounds, N-acryl substituted (meth) acrylamide derivatives, N, N-dialkyl substituted Polymers or copolymers such as (meth) acrylamide derivatives, N-heterocyclic group-substituted (meth) acrylamide derivatives, and vinyl ether derivatives are used. Moreover, complex formation can also be utilized like polymethacrylic acid-polyethylene glycol.
[0013]
In the case of photosensitivity, for example, polyhydroxyethyl methacrylate having an azobenzene group, which is a combination of a dye that causes photoisomerization and a hydrogel, a vinyl derivative of triphenylmethane leuco hydroxide and an acrylamide compound. An ion gel having a sensitive group capable of photoion dissociation, such as a polymer, or an N-isopropylacrylamide gel containing spirobenzopyran, in which hydrophobic interaction is photochanged, is used.
[0014]
In the case of electrosensitivity, for example, an ionic dissociation group is formed like a hydrolyzed part of acrylamide gel or polymethacrylic acid or 2-acrylamido-2-methylpropanesulfonic acid-2-hydroxyethyl copolymer. Something you have used is used.
[0015]
In addition, in the case of a gel that responds to both light and electricity, such as an ion gel having a sensitive group that undergoes photoion dissociation, both stimuli can be used.
[0016]
In addition, when adjusting the affinity between blood cells and gel, or when it is necessary to adjust the interaction between the polymer gel and the porous substrate or fiber assembly, in order to change the properties of the polymer gel, Copolymerization with monomers other than those described above, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. It is also possible to crosslink within a range that does not impair the original properties of the polymer.
[0017]
The method of coating these gels on the polymer porous body or fiber assembly can be used alone or in combination with a chemical reaction or a physical interaction. Examples of the method of bonding by chemical reaction include electron beam irradiation, gamma ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, or radical reaction, anion when the substrate and the gel have appropriate reactive functional groups. Commonly used organic reactions such as reactions and cation reactions can be used. As a method utilizing physical interaction, for example, there is a method using physical adsorption such as coating or kneading using a gel alone or a matrix having good compatibility with a substrate as a medium.
[0018]
The cell separation material obtained in this manner is a module such as a flat membrane or a hollow fiber membrane, a porous mass, a column containing an aggregate of fibers, a nonwoven fabric or a woven fabric, depending on the form of the substrate. Or it accommodates in containers, such as a module which accommodated knitting, and is used as a cell separation filter. The form of the container is easy to use a fiber column, fiber filter, or porous body filter that is generally used for leukocyte removal filters, but any form that is suitable for separating and collecting the target blood cells can be used. I do not care.
[0019]
The cell separation filter has an inlet and an outlet for blood containing a target blood cell. In addition, the blood inlet and outlet may be the same. Further, an outlet for blood cells to be separated and recovered may be provided separately, or a port for introducing a rinsing solution such as physiological saline may be provided. Furthermore, a device for providing a signal corresponding to the responsiveness of the cell separation material such as temperature, light, electricity, etc. is provided near the cell separation filter. Here, the circuit that contacts the blood and the cell separation filter are desirably disposable, but it is desirable that the device for providing these signals be integrated with a holder or the like that supports the cell separation filter in order to miniaturize the device.
[0020]
A blood cell separation system including this cell separation filter can be introduced between blood storage bags to perform a separation operation when transferring blood from the bag to the bag. It can also be used in combination with a leukocyte removal filter or plasma separator. In addition, it can be used for platelet pheresis or leukocyte pheresis in combination with a blood collection circuit, an anticoagulant addition circuit, and a blood cell storage bag.
[0021]
[Action]
The operation of the present invention will be described with reference to an example in which platelets are separated and collected by a filter using a temperature sensitive gel.
[0022]
When introducing a liquid containing platelets into the filter, the N-propylacrylamide gel is kept at 40 ° C. in a contracted state. In general, since the gel is more hydrophobic in the contracted state than in the swollen state, the platelets adsorb to the gel on the surface of the filter medium. In this case, the gel is adjusted so that the platelet is adsorbed to such an extent that it does not deform to such an extent that it loses its function. After all the liquid to be treated is filtered through a filter, the temperature is set to 30 ° C. so that the gel swells. When the gel swells, it becomes hydrophilic, the interaction with platelets weakens, and platelets elute. At this time, if necessary, an appropriate eluent may be introduced into the filter.
[0023]
According to this method, the operation is not complicated and time-consuming as in the case of centrifuging the bag, and high-purity platelets are obtained without using a large and expensive device such as a blood separation device. I can do it. In addition, leukocytes can be separated and collected by the same method, and high purity and recovery rate can be obtained as compared with the conventional method in which the affinity between the filter medium and leukocytes is not changed. Furthermore, if the difference in the affinity of platelets and leukocytes for the filter medium is used, there will be one state change due to a three-stage state change: a state in which both have a high affinity, a state in which only one has a high affinity, and a state in which both have a low affinity. It is also possible to separate and collect two types of blood cells with this filter.
[0024]
【Example】
A polyurethane porous membrane having a thickness of 7.2 mm and an average pore diameter of 3 μm was mixed with an N-propylacrylamide aqueous solution, dried, and then irradiated with gamma rays to form a water-insoluble gel. This membrane was punched out into a circle with a diameter of 47 mm and sandwiched between filter holders, and a PVC tube was connected to the filter entrance. With the filter placed in a constant temperature bath at 40 ° C., 50 ml of human stored blood (with CPD) was fed at a rate of 0.6 ml / min with a roller pump, and the blood that came out of the filter was collected. After all the blood was collected, the filter was placed in a thermostatic bath at 25 ° C., and 50 ml of physiological saline was then fed at the same speed, and the liquid coming out of the filter was collected.
[0025]
The number of platelets in human stored blood before filtration, human stored blood after filtration, and physiological saline that passed through the filter was 230,000, 220,000, and 186,000 (pieces / microliter), respectively.
[0026]
【The invention's effect】
In the present invention, high cell separation / recovery could be performed by simple operations such as filtration / rinsing and temperature stimulation adjustment.

Claims (1)

It consists of a polymer porous body or a fiber assembly coated with a polymer gel whose affinity to platelets and leukocytes changes in response to external stimuli, and uses the difference in the affinity of platelets and leukocytes to the filter medium A material for separating platelets or leukocytes from blood or blood products .