JPS6139060B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a monocyte/granulocyte capture/collection filter.

More specifically, the present invention relates to a filter that can capture and collect monocytes and granulocytes from blood, body fluids, or a blood cell suspension obtained by processing these in a short time with simple operations.

In recent years, with advances in hematology and immunology, blood component transfusions have been developed to replace conventional whole blood transfusions, such as granulocyte transfusion, leukocyte surface antigen testing using lymphocytes, and lymphocyte subpopulation testing. It performs ratio measurements and is applied to the treatment and diagnosis of various diseases. Furthermore, attempts to classify and separate cells into sublets such as helper T cells and suppressor T cells are beginning to be carried out in hospitals and research institutes throughout the country.

Conventional techniques for capturing (removing) and collecting monocytes and granulocytes that can be used for this purpose include centrifugation, density gradient centrifugation, and methods for adhering monocytes and granulocytes to fibers. be.

More specifically, the centrifugation method involves centrifugation to obtain a fraction rich in white blood cells, and the density gradient centrifugation method involves layering blood in a liquid with a specific gravity of 1.077, followed by centrifugation to collect the lymphocyte layer. The method of adhering monocytes and granulocytes to fibers involves flowing blood through a column filled with fibers such as nylon or glass wool to capture the granulocytes, and then recovering the granulocytes captured in the column. how to do it,
A leukocyte-rich fraction is obtained using a hemagglutinating agent and a centrifuge, and then this leukocyte fraction is transferred to nylon,
This method involves placing the column in a column filled with glass wool, keeping it warm at 37°C, leaving it for about 30 minutes, and then collecting the lymphocytes.

However, these methods had the following drawbacks. That is, the white blood cell fraction obtained by the centrifugation method contains many lymphocytes in addition to monocytes and granulocytes. In the density gradient centrifugation method, the resulting lymphocyte layer is often contaminated with monocytes and platelets, and in order to remove these, a thrombin solution is added to aggregate the platelets and monocytes, and multiple centrifugal washes are required. In addition, many lymphocytes are lost during these operations, and the operations are complex and time-consuming, making it difficult to perform aseptic operations, and the apparatus itself is expensive. In the method using fibers, the process of creating a column uniformly packed with fibers was difficult and time-consuming.

Therefore, the present inventors discovered that blood can be easily extracted from blood.
As a result of intensive research aimed at capturing (removing) and collecting monocytes and granulocytes with high purity and high yield in a short time, we found that the average pore diameter was 25 to 60 micrometers (μ
The porous filter (m) selectively captures monocytes and granulocytes, and the captured monocytes and granulocytes can be recovered with a simple operation, and since the porous filter is the main part, they can be sealed in a container. The inventors have also found that the method is simple, leading to the present invention.

That is, the present invention is a filter for trapping and collecting monocytes and granulocytes, the main part of which is a porous filter having continuous pores with an average pore diameter of 25 to 60 μm.

Any material can be used for the porous filter as long as it does not cause damage to blood cells.
Synthetic rubber, synthetic resin foam, etc. are easy to use as the pore diameter can be easily adjusted.

The present invention will be explained below using the drawings. FIG. 1 is a schematic cross-sectional view of a porous filter used in the present invention. The porous filter used in the present invention refers to a filter in which the pores 1 are randomly opened as shown in FIG. 1, and the pores are continuous from the surface of the porous structure 2 to other surfaces. Even though it is porous, it does not necessarily have to be flexible. The average pore diameter is calculated by cutting a porous filter arbitrarily, measuring the diameter of each pore dispersed throughout the cross section, and examining the relationship between the diameter and the number of pores.
It represents the diameter converted into a circle of the largest number of pores. In other words, the pores dispersed on any cut surface of a porous filter have various shapes and diameters, but each pore is converted into a circle with the same area as the cross-sectional area of the pore. If you draw a graph with the diameter on the horizontal axis and the number of pores on the vertical axis, you will generally get a curve close to a normal distribution. At this time, it is preferable to randomly count 1000 or more pores. The diameter corresponding to the peak of the curve is the average pore diameter in the present invention. Therefore, when various particles are passed through a porous filter, particles with a diameter larger than the average diameter of the porous filter are difficult to pass through, and particles with a diameter larger than this are said to never pass through. It's nothing.

Further, it is desirable that the porous filter has a higher pore porosity than a porous structure, and it is also desirable that the pore size distribution of the pores be narrower.

The following is an example of how the present invention captures and captures monocytes and granulocytes.
Describe the sampling filter. Fig. 2 is a schematic diagram showing an example of the monocyte/granulocyte capture/collection filter of the present invention, and Fig. 3 is an example of an apparatus for using the monocyte/granulocyte capture/collection filter of the present invention. FIG.

The filter for capturing and collecting monocytes and granulocytes of the present invention includes, for example, a container 5 having an inlet 3 and an outlet 4 as shown in FIG.
A porous filter 6 is housed inside. When this filter is actually used, for example, an experimental apparatus as shown in FIG. 3 is used. Taking as an example an experiment to remove monocytes and granulocytes from blood, blood 7 is first sent to a monocyte/granulocyte capture/collection filter 9 by a pump 8, where most of the monocytes and granulocytes are removed. Most of the lymphocytes, red blood cells, plasma, etc. are not captured by the filter 9 and are sent to the container 10.

The reason why monocytes and granulocytes are selectively captured by Filter 9 is that monocytes and granulocytes have higher adhesiveness than other blood cells, while lymphocytes and red blood cells have lower adhesiveness. In particular, since red blood cells have high deformability, they are hardly captured by the filter 9.

In this way, by using the monocyte/granulocyte capture/collection filter of the present invention, monocytes/granulocytes can be selectively and efficiently captured (removed) from blood in a short time with simple operations. Furthermore, by adopting an appropriate collection method for monocytes and granulocytes captured by the monocyte and granulocyte trapping/collection filter, it is now possible to recover them with good purity and yield. Summer.

Here, the average pore diameter of the porous filter is in the range of 25 to 60 μm, preferably 30 to 50 μm in terms of both selectivity and efficiency. Average pore size is 25μm
If the concentration is below, even lymphocytes with low adhesiveness will be easily captured by the filter, making it difficult to selectively capture (remove) only monocytes and granulocytes. Furthermore, when the average pore size is 60 μm or more, it becomes difficult for monocytes and granulocytes to adhere to the filter, making it difficult to use it as a filter for trapping and collecting monocytes and granulocytes.

In addition, in order to prevent excessive leakage of blood cells to be captured due to non-uniformity of the pore diameter of the porous filter, the thickness of the porous filter, that is, the linear distance from the blood inlet to the outlet of the porous filter, is set to 10 mm.
The above shall apply.

Example 1 Polyester foam with an average pore size of 50 μm was
It was placed in a 20 mm x 75 mm long container and used as a filter for capturing and collecting monocytes and granulocytes. The experimental apparatus shown in FIG. 3 was used. Add 4ml/50ml of heparinized blood from a healthy person to this filter.
When the blood that came out as a liquid was examined by flowing it at a flow rate of min., it was found that monocytes and
The concentration of granulocytes was only 3%, whereas the concentration of lymphocytes was 80% and the concentration of red blood cells was approximately 100%. After this, pour physiological saline into the filter at a flow rate of 4.
ml/min to wash the blood remaining on the filter, and when 100 ml of liquid was obtained, 50 ml of physiological solution containing plasma was flowed through the filter at a flow rate of 5 ml/min.
Monocytes and granulocytes inside the filter were collected while applying vibration to the filter. When the collected fluid was examined, it was found that 60% of the original blood contained monocytes and granulocytes, 6% lymphocytes, 0.2% red blood cells, and 4% platelets.

Example 2 A polyether urethane foam material with an average pore size of 30 μm was placed in a container with an inner diameter of 10 mm and a length of 50 mm and used as a filter for trapping and collecting monocytes and granulocytes. Through this filter, 5 ml of a leukocyte suspension obtained by resuspending the leukocyte fraction obtained from healthy human heparinized blood by centrifugation in plasma was kept at 37°C and flowed at a flow rate of 1 ml/min. It was flowed at a flow rate of min to obtain 5 ml of liquid. When the white blood cells in this fluid were examined, the ratio of monocytes/granulocytes to lymphocytes was 6:94, that is, lymphocytes were obtained with a purity of 94%. The recovery rate for lymphocytes in the leukocyte suspension was 65%.

As mentioned above, the present invention can capture and capture monocytes and granulocytes.
By using a collection filter, it is possible to selectively and efficiently capture (remove) monocytes and granulocytes from blood in a short time with a simple operation. It has also become possible to collect granulocytes with high purity and high yield. Also,
Since the main part is a porous filter, it is very easy to seal it in a container.

[Brief explanation of the drawing]

FIG. 1 is a schematic diagram showing a cross section of a porous filter according to the present invention, FIG. 2 is a schematic diagram showing an example of a filter for capturing and collecting monocytes and granulocytes according to the present invention, and FIG. 3 is a schematic diagram showing a cross section of a porous filter according to the present invention. - It is a schematic diagram showing an example of a device when using a granulocyte capture/collection filter. 1... Pore, 2... Porous structure, 3... Inlet, 4... Outlet, 5... Filter container, 6...
... Porous filter, 7... Blood, 8... Pump, 9... Monocyte/granulocyte capture/collection filter, 10... Container.

Claims (1)

[Claims] 1. The average pore diameter is from 25 to 60 micrometers (μ
m) A filter for capturing and collecting monocytes and granulocytes, the main part of which is a porous filter having continuous pores. 2. Capture of monocytes and granulocytes according to claim 1, wherein the porous filter has a thickness of 10 mm or more.
Collection filter.