JPS6252620B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a separation material that adheres to granulocytes and monocytes and separates lymphocytes from a leukocyte suspension. More specifically, the present invention relates to a separation material that is a synthetic resin gel-type granular hydrophobic solid material containing an acidic functional group. The concentration of acidic functional groups is 1.2 m equivalent (millichemical equivalent) per 1 ml volume of the wet material, and the particle size of the wet material is 30 μ or more.
This relates to a separation material made of 350μ or less. Conventionally, methods for removing granulocytes and monocytes from leukocyte suspensions and isolating lymphocytes are based on the differences in density, size, and morphology between the two cells, as well as the phagocytosis and adhesive ability of granulocytes and monocytes. Methods that utilize properties and methods that combine and separate these are known. On the other hand, with recent remarkable developments in cellular immunology, it is important to influence as much as possible the ratio of lymphocytes to their sub-fractions, T cells (thymus-derived lymphocytes) and B cells (bone marrow-derived lymphocytes). Without,
Moreover, there is an increasing need to quantitatively separate leukocytes from leukocyte suspensions, ie, with high yield and high purity, and without impairing lymphocyte function. However, in conventional separation methods, there are overlapping fractions between lymphocytes, granulocytes, and monocytes, resulting in poor quantitative performance and often causing cell damage. For example, even if lymphocytes are separated by specific gravity centrifugation using sodium metrizoate ficoll, which is the most commonly used method, although granulocytes can be removed, the same molecule as lymphocytes may be removed. Monocytes, which are nuclear cells, are mixed in. Furthermore, when leukocytes from cancer patients are used, granulocytes often change in density and become contaminated. In order to remove these contaminating monocytes and granulocytes, a combination of methods has been used to remove them by feeding them with Diaionenacarbonyl particles and latex particles.
Cell damage to lymphocytes is unavoidable, and some B cells, a sub-fraction of lymphocytes, are often lost. Furthermore, the performance of separation methods that utilize differences in adhesion to fibers is greatly affected by fiber spacing and fiber packing density, and not only is it difficult to obtain stable performance, but in many cases it is difficult to obtain highly pure lymphocytes. If you try to get some sticky B cells,
Furthermore, some T cell subfractions tend to be lost. In addition, methods for separating polysaccharides with a certain particle size, such as agarose gel or dextran gel, using differences in their adhesion to gels,
It lacks thermal and chemical stability, is prone to microbial growth, has difficulty maintaining separation performance, and furthermore, the gel itself tends to stick to some B cells. As is clear from the above, it is possible to isolate lymphocytes using a leukocyte suspension as a specimen with simple manipulations, with high purity and recovery rate, and without causing cell damage. Although it is widely expected in the fields of medicine and biology, with a focus on science, the current situation is that it has not been achieved. The present inventors have made extensive efforts to develop selection-specific cell separation materials with the aim of easily separating lymphocytes with high purity and recovery rate without damaging cell membranes and cell functions using a leukocyte suspension. As a result of repeated research, we found that the concentration of acidic functional groups is 1.2 m equivalent or more per 1 ml volume of wet material, and the particle size of wet material is 30 μ or more.
By contacting a gel-type particulate hydrophobic solid substance made of a synthetic resin containing an acidic functional group with a size of 350μ or less and a white blood cell suspension in a protein-containing liquid, the substance (hereinafter abbreviated as the separation material of the present invention) is produced. The present invention was completed based on the discovery that the present invention does not adsorb lymphocytes. That is, by contacting the separation material of the present invention with a leukocyte suspension in a protein-containing solution, it was found that the negative charge on the cell membrane of lymphocytes is greater than that of granulocytes and monocytes, and that the adhesiveness of the cells is reduced to granulocytes. Since the additive effect of globules and monocytes being stronger than lymphocytes can be fully exerted, lymphocytes do not adhere to the separation material of the present invention, and only granulocytes and monocytes adhere. , becomes separable. Therefore, a column having a liquid inlet/outlet and one or more filters is prepared, filled with the separation material of the present invention immersed in a liquid containing 1 g/dl or more of protein, and 1 g of blood or leukocytes separated from blood is prepared. By injecting a suspension of protein into the column and washing it out with a washing solution, a solution in which only lymphocytes are selectively suspended can be obtained. In the present invention, a particulate hydrophobic solid material containing an acidic functional group can be defined as a particulate hydrophobic solid material in which an acidic functional group, that is, a strongly acidic functional group or a weakly acidic functional group, is present. The strong acidic functional group is, for example, a sulfone group, and the weakly acidic functional group is, for example, a carboxyl group, a phosphonic group, a phenol group, etc. Specifically, a copolymer of styrene and divinylbenzene with each of the above acidic functional groups directly bonded, for example, 4 to 12% divinylbenzene is added to styrene, and a small amount of peroxide is added to the mixture as a polymerization catalyst. It is obtained by adding benzoyl and water, adding a suspending agent such as bentonite or alginic acid to carry out suspension polymerization, and treating the resulting polymer with concentrated sulfuric acid or chlorosulfonic acid. In addition, condensates of phenolsulfonic acid, formaldehyde, and phenol, vinylsulfonic acid polymers, acrylic acid polymers, methacrylic acid polymers, itaconic acid polymers, maleic acid polymers, fumaric acid polymers, butadiene-1- Examples include carboxylic acid polymers, iminodiacetic acid type styrene polymers, condensates of 1,3,5-resorcinic acid and formaldehyde, diallylphosphonic acid polymers, and condensates of phenol and formaldehyde. The concentration of acidic functional groups in the present invention is suitable for exhibiting performance if it is 1.2 m equivalent or more per 1 ml of wet volume, and if it is less than 1.2 m equivalent, the lymphocyte adsorption rate to the separation material will decrease. is on the rise. There is no particular upper limit to the concentration of acidic functional groups, but those with an excessive concentration of acidic functional groups are
Cannot be used due to strength deterioration. The acidic functional group is H
form (e.g. -SO ₃ H) or salt form [e.g. -
[SO ₃ Na, −SO ₃ K, (−SO ₃ ) ₂ Ca, (−SO ₃ ) ₂ Mg, etc.] can be used, but the salt form is preferred because it is easier to adjust the pH of the suspended liquid. preferable. The particle size in the present invention is suitably 350μ or less, preferably 200μ or less, in order to exhibit its performance in the wet state of actual use, that is, in a wet state, and if it is larger than 350μ, The adsorption rate of granulocytes and monocytes to the separation material tends to decrease. In terms of wet particle size, in a particle size distribution that can be considered to be a normal distribution, the separation material has a particle size of 90%.
30μ or more 350μ as effective diameter including % or more
Particles within the following particle size range, preferably from 70μ to 200μ, can be used, and particles having an effective diameter within this range may be appropriately mixed and used. An example of a separator for separating lymphocytes used in the present invention will be described with reference to a drawing. A column 3 has an inlet 1 for injecting a cell suspension and a washing solution, and an outlet 2 for draining the cell suspension and washing solution.
At the outlet, a filter 4 having a pore size large enough to prevent the separation material from flowing out is attached, and the column 3 is filled with the separation material 5 described above. To separate lymphocytes from white blood cells using a separator, white blood cells separated from blood or blood by a method such as centrifugation or specific gravity centrifugation are suspended in a protein-containing solution and then washed with a protein-containing solution in advance. When the suspension liquid has permeated into the separation material of the present invention, a washing liquid is poured to wash out unabsorbed cells. The washing liquid washed out by the above method contains lymphocytes. The protein-containing liquid used when bringing the separation material of the present invention into contact with leukocytes includes a liquid containing 1 g/dl or more of protein, preferably autologous serum or animal serum.
For example, a culture solution or buffer containing 30% or more fetal bovine serum or calf serum, more preferably 100% serum, is used. When a solution containing less than 1 g/dl of protein is used, for example when a buffer solution containing 10% serum is used, the rate of adsorption of lymphocytes to the separation material tends to increase. In addition, if the separation material is coated in advance with a solution containing 1 g/dl or more of protein, there is no need to wash the separator with a protein-containing solution beforehand, making the separation process easier. It's convenient. The temperature during the separation operation may be any temperature as long as it does not cause cell damage, but room temperature (18-25℃) is acceptable.
The preferred temperature is from to body temperature (around 37 degrees Celsius). The lymphocytes obtained in this way were subjected to immunological function tests such as blastogenesis ability, antibody production regulation effect, cytotoxicity ability, and antibody production ability. The function was maintained to the same extent as the function of . Furthermore, after eluating the lymphocytes, 1
By gently stirring the separation material while adding a solution containing the same protein used in the separation of g/dl or more, the once adsorbed leukocytes can be eluted and recovered. The leukocytes thus obtained have excellent viability, and no particular decrease in function is observed in immunological function tests. The above is a particulate hydrophobic solid substance containing an acidic functional group, in which the concentration of the acidic functional group is 1.2 m equivalent or more per 1 ml volume of the wet material, and the particle size of the wet material is 30 μ or more.
A separation material for adsorbing granulocytes and monocytes from a leukocyte suspension and separating lymphocytes, which is characterized by a particle size of 350 μ or less, and a separation material for separating lymphocytes from a leukocyte suspension;
The present invention, which separates lymphocytes from a leukocyte suspension by contacting them in a solution containing protein of g/dl or higher, has the following effects compared to conventional lymphocyte separation samples and methods thereof. (1) The procedure for separating lymphocytes from white blood cells is simple and can be completed in one step. That is, all that is required is to inject the leukocyte suspension into a container filled with the separation material of the present invention and to drain the washing liquid. (2) There is no need for enzymatic or drug treatment of leukocytes during separation, and the properties of the lymphocytes do not differ from the original lymphocytes even after separation, so no functional changes or declines in lymphocytes occur. Therefore, it can also be used for immunological function tests on lymphocytes. (3) The production of the separator is easier and cheaper than the production of conventional lymphocyte separation samples and separation equipment. (4) Since the separating material is a polymeric synthetic material, it is easy to produce a separating material with a certain level of activity and maintain the activity, and the sterilization process can be easily performed. Furthermore, it has excellent physical, chemical, and biological stability,
There is no deterioration over time. Next, the present invention will be explained in more detail with reference to Examples. Example 1 Preparation of separator: 2 ml of gel-type granular sulfonated polystyrene-divinylbenzene resin of Na ion type (acidic functional group concentration: 1.5 m equivalent per 1 ml volume of wet material, particle size of wet material: effective diameter 100 to 350 μ) After thoroughly immersing the resin in phosphate buffer and washing, the resin is
Inner diameter 10 with a liquid inlet and outlet with a 50 μm mesh nylon net filter on the bottom.
It was prepared by filling a 2 mm acrylic plastic column (see drawing), taking care not to introduce air bubbles. Separation procedure: Sodium metrizoate-ficoll mixture (d=
1.077, 20°C), the leukocyte fraction was separated by specific gravity centrifugation and washed with phosphate buffer.
Gently add 0.5 ml of a solution suspended in 100% fetal bovine serum (protein content: 4.5 g/dl) to a concentration of 10 ⁶ /ml from the inlet of a separator that was previously washed with 4 ml of 100% fetal bovine serum. When the floating liquid has sufficiently permeated the separation material, immediately pour 4 ml of fetal bovine serum from the inlet of the column as a washing solution to wash out cells that are not adsorbed to the separation material at a flow rate of gravity. Recovered. Analysis results: Total cell recovery rate was determined by microscopic observation using an automatic hemocytometer or hemocytometer. In addition, the lymphocytes in the specimen and collected solution were analyzed by analyzing the sub-fractions T cells by rosette reaction with neuraminidase-treated sheep red blood cells, and B cells by fluorescent antibody method against surface immunoglobulin. Calculated by the sum of
On the other hand, granulocytes and monocytes in the specimen and recovered solution were analyzed by peroxidase staining.
Furthermore, the recovery rate of each composition was calculated using the total cell recovery rate and the proportion of each composition in the specimen and recovery solution. The results are shown in Table 1, including data on T cells and B cells. Examples 2 to 5 By the same method as in Example 1, gel-type granular sulfonated polystyrene-divinylbenzene resin was prepared.
Na ion type (acidic functional group concentration: 2.0 m equivalent per 1 ml wet volume, wet particle size: effective diameter 70 to 300μ)
-Example 2-, Na ion type of gel-type granular methacrylic acid-divinylbenzene resin (acidic functional group concentration: 2.5 m equivalent per 1 ml volume of wet body, particle size of wet body: effective diameter 100 to 350μ) - Example 3 -, Na ion type of gel type granular acrylic acid-divinylbenzene resin (acidic functional group concentration: 3.5 per ml wet volume)
m equivalent, wet particle size; effective diameter 70 to 350μ) - Example 4 - Gel type granular carboxylated polystyrene -
The results of experiments using Na ion type divinylbenzene resin (acidic functional group concentration: 4.1 m equivalent per 1 ml wet volume, wet particle size: effective diameter 70 to 350μ) - Example 5) are shown below. They are summarized in Table 1.

[Table] Comparative Examples 1 and 2 Using a leukocyte suspension with the same concentration and composition as in Example 2, gel-type granular sulfonated polystyrene
Na ion type of divinylbenzene resin (acidic functional group concentration: 1.0 m equivalent per 1 ml wet volume, wet particle size: effective diameter 70 to 350μ) - Comparative Example 1 - Gel type granular sulfonated polystyrene - divinylbenzene resin Na ion type (acidic functional group concentration: 2.0 m equivalent per 1 ml wet volume, wet particle size: effective diameter 380 ~
550μ) - Comparative Example 2 - were used as separation materials, respectively.
The same separation operation as in Example 1 was carried out. The analysis results are summarized in Table 2. Comparative Example 3 A leukocyte suspension with the same concentration and composition as in Example 2 was used, except that 10% fetal bovine serum-phosphate buffer (protein content 0.44 g/dl) was used instead of 100% fetal bovine serum. The experiment was carried out in the same manner as in Example 1. The analysis results are shown in Table 2.

[Table] Example 6 Na ion type of gel type granular sulfonated polystyrene-divinylbenzene resin (acidic functional group concentration;
Using 1.8 m equivalent per 1 ml volume of wet fluid (particle size of wet fluid; effective diameter: 70 to 200 μ) as a separation material, the buffer coat was separated from heparinized human peripheral blood by centrifugation, and then thoroughly separated with phosphate buffer. The procedure was carried out in the same manner as in Example 1, using 0.5 ml of the washed leukocyte fraction suspended in 100% fetal bovine serum at a concentration of 1×10 ⁷ /ml as the sample solution. As a result of the analysis, the composition ratio in the sample fluid was lymphocytes = 38.5% (T = 35.2%, B = 3.3%) and granules/monocytes = 61.3%, whereas the composition ratio in the collected fluid was Lymphocytes = 79.1% (T = 72.4%, B = 6.7
%), granules/monocytes = 19.1%, and the recovery rate was total cells = 45.5%, lymphocytes = 93.5% (T = 93.6%,
B = 92.4%), and granules/monocytes = 14.2%. Example 7 Using the same separation material as in Example 6, leukocytes were isolated.
The experiment was carried out in the same manner as in Example 1, using 0.5 ml of heparinized human peripheral blood containing a concentration of 7.86×10 ⁶ /ml as the sample fluid. As a result of the analysis, the composition ratio in the sample fluid was lymphocytes = 37.1% (T = 27.5%, B = 9.6%) and granules/monocytes = 62.0%, whereas the composition ratio in the collected fluid was Lymphocytes = 85.2% (T = 64.8%, B = 20.4
%), granules/monocytes = 12.6%, and the recovery rate was total cells = 36.3%, lymphocytes = 83.4% (T = 85.5%,
B = 77.1%), and granules/monocytes = 7.4%. The recovered solution also contains red blood cells and platelets, so if you want to remove them,
Furthermore, hemolysis and centrifugation operations are required. Example 8 A separator was created in the same manner as in Example 1, and after washing out unadsorbed cells using the same separation procedure, 8 ml of phosphate buffer containing 10% fetal bovine serum was poured into the column from the inlet. The separation material is gently stirred by pipetting with a Pasteur pipette while flowing down at the flow rate of gravity, and the separated white blood cells are eluted. The obtained leukocytes were analyzed in the same manner as in Example 1, and the composition ratio was 23.6% T lymphocytes, 10.7% B lymphocytes, and 64.3% granules and monocytes.

[Brief explanation of the drawing]

The drawing is a sectional view showing one example of a separator. 1...Liquid inlet, 2...Liquid outlet, 3...Column, 4...Filter, 5...Separation material.

Claims (1)

[Claims] 1 Gel-type granular hydrophobic solid material made of synthetic resin containing acidic functional groups, in which the acidic functional group concentration is 1.2 m equivalent or more per 1 ml of wet material, and the particle size of the wet material is 30 μ or more and 350 μ or less A separation material that adheres to granulocytes and monocytes and separates lymphocytes from a leukocyte suspension.