JP2004101256A - Method for screening biomaterial - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening biomaterials for cell medical services. <P>SOLUTION: The screening method comprises a series of the following methods. A cell of which the interaction with the biomaterials is to be measured is determined, and lectin which specifically combines with the cell is selected. Then, a probe for screening the biomaterials containing a sugar chain having an affinity to the lectin is selected. By measuring the interaction between the probe for screening the biomaterials and a candidate material, a target biomaterial is finally selected. The biomaterial is useful as a material for medical equipment such as a leukocyte removal filter for blood transfusion. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for screening a desirable biomaterial when a material such as a synthetic polymer is used as the biomaterial. The biomaterial selected according to the present invention can be used in the manufacture of medical devices and medical devices, including a leukocyte removal filter for blood transfusion, and can also be used in the field of regenerative medicine such as a cell storage bag, a cell separation membrane, and a cell incubator.
[0002]
[Prior art]
Various medical devices such as a hemodialyzer, a leukocyte adsorption removal filter for blood transfusion, and an artificial blood vessel are made of a so-called biomaterial. The interaction between the biomaterial and the biological substance is important, and a more appropriate one must be selected according to the properties required for the biomaterial. In recent years, the properties required for these various medical devices have become more sophisticated, and research and development of new materials has been actively pursued. If you must use, there are a number of significant challenges besides ethical issues. For example, in the case of a leukocyte removal filter for blood transfusion, first, it is extremely difficult to obtain large quantities of blood products essential for medical treatment. Second, even if available, blood products do not always contain the same amount of components and the number of days they have been stored, so individual differences between blood products are large, and this individual difference fluctuates the results of material evaluation. In addition, different methods of preparing blood products in different countries and facilities may contribute to individual differences in blood products. Third, pathogens such as syphilis, hepatitis (types B and C) and AIDS have been tested, and negative ones are blood products. However, unknown pathogens and so-called window The danger still exists.
[0003]
Various attempts have been made to overcome these problems. For example, Patent Literature 1 discloses a method for evaluating the performance of a leukocyte removal filter without using blood by using simulated blood in which particles (artificial products) having a specific particle size are dispersed in a liquid having a specific viscosity. Have been. However, although this method certainly correlates with the evaluation using blood, it is a very indirect and limited method that has no theoretical support and, of course, is not applicable to anything other than blood cells . In the final stage of medical material development including actual clinical experiments, it is necessary to examine the actual scale of whether or not the product specifications are satisfied. However, up to that point, experiments will be conducted on the smallest possible scale. However, if the technique uses blood even in a small amount, the above-mentioned first and second problems may be solved, but the third problem, infection, cannot be solved. These challenges, even when using non-human xenogeneic cells, are the first quantitative challenge, and the third, including the recent xenogeneic infections such as mad cow disease, It remains a major obstacle to biomaterials research and development.
[0004]
By the way, the outermost surface layer of the eukaryotic cell membrane is composed of a layer containing a large amount of sugar called "sugar coating", and these sugars exist as glycoproteins and glycolipids by covalently bonding with membrane proteins and lipids. Some exist as polysaccharides in the integral membrane proteoglycan molecule. In recent years, advances in research on glycotechnology have been remarkable, such as pathological changes caused by abnormal carbohydrate-receptor interactions and the role of sugar chains in viral infections such as AIDS, and lectins have been used as tools for these studies. That is noteworthy. Lectins that exhibit specific binding to sugar chains are mainly derived from plants, and over 100 types of lectins have been found so far, and many of them are commercially available as sugar chain research reagents. Regarding the use of lectins, for example, Non-Patent Document 1 discloses that soybean agglutinin, which is a kind of lectin, has high affinity for more differentiated cells. A cell separation method for enrichment is shown. For example, Patent Document 2 discloses a substrate used for cell separation or the like by immobilizing lectin, and a method for immobilizing lectin. This is essentially different from the gist of the present invention in that a lectin is used to select a probe for biomaterial screening.
[0005]
[Patent Document 1]
JP-A-11-76396 [Patent Document 2]
JP-A-8-319300 [Non-Patent Document 1]
Exp. Hematol 1994, 22 (12): 1134-40.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to eliminate the risks of (1) quantity limitation, (2) individual difference or lot difference, (3) infection risk and to screen biomaterials for cell medicine, which are problems when actually using cells. And a biomaterial screened in this way.
[0007]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve such problems, and first focused on the fact that when a biomaterial interacts with a cell, the sugar chain present on the outermost surface of the cell is directly affected. He hypothesized that a substance containing these sugar chains on the surface could substitute for cells. Next, using conventional lectin-based cell research methods to identify sugar chains on the surface of the target cell membrane, and whether the substances containing the specific sugar chains of these high-binding-affinity lectins can substitute for cells And completed the present invention.
[0008]
That is, the present invention provides (1) a cell for which the interaction with the biomaterial is to be measured, (2) a lectin that specifically binds to the cell, and (3) a saccharide having an affinity for the lectin. Four steps of selecting a biomaterial screening probe including a chain, (4) measuring the interaction between the biomaterial screening probe and the candidate material, and screening the biomaterial from the candidate material based on the measurement result. And a biomaterial selected by the method.
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
The biomaterial as referred to in the present invention is a material that constitutes the above-mentioned medical device and medical device, such as cell collection, separation, storage, and culture.
[0010]
The lectin as referred to in the present invention is a protein that specifically binds to sugar and binds to a sugar chain of a cell membrane glycoconjugate (glycoprotein or glycolipid) to induce cell aggregation, mitogenesis, activation of function, and cytotoxicity. It is a substance generally known as a substance having such an effect. Typical ones are Gingerbread lectin, mushroom lectin, concanavalin A, doricos bean lectin, Korean Asaga lectin, deigo bean lectin, lentil lectin, lotus lectin, dog lentin lectin, kidney bean lectin, peanut lectin, pea lectin, United States There are pokeweed lectin, pokeweed lectin, soybean lectin, Japanese elder lectin, gorse lectin, wheat germ lectin and the like.
[0011]
In addition to tissue staining, enzyme immunity and flow cytometry can be used to detect the affinity between cells and lectins. The lectins used are labeled in advance with biotin, Fluorescein Isothiocyanate (FITC), Horse Radish Peroxidase, or the like. Labeled lectins can be used.
[0012]
The first step of the present invention is to use lectins to characterize cell surface properties resulting from the interaction with the biomaterial. A probe for biomaterial screening is selected from information on the sugar specificity obtained in this step, and is used in the next step. The cell surface properties characterized by using lectins refer to the type and number of sugar chain molecules. For example, Hilochawantake lectin is α-L-Fuc, mushroom lectin is β-D-Gal, and concanavalin A is α-D-Man, α-D-Glc, Drico's bean lectin is D-GalNAc, Datura asaga lectin is β-D-GlcNAc, deigo bean lectin is D-GalNAc or D-Gal, lentil lectin is D-Man or D-Glc, Lotus lectin is L-Fuc, canine endectin is Siaα2-3Gal, kidney bean lectin is D-GalNAc, peanut lectin is D-Gal, pea lectin is D-Glc or D-Man, and American pokeweed lectin is chitooligo. Sugar and bean lectin are β-D-Gal, Down the D-GalNAc, Nihon elder lectins Siaα2-6Gal / GalNAc, Hari broom lectins α-L-Fuc, wheat germ lectin is D-GlcNAc are known as the main sugar specificity.
[0013]
In order to characterize the cell surface using lectins, cells are reacted with various lectins, and the reaction conditions at that time are desirably adjusted to the intended use form of the biomaterial. For example, for the purpose of screening a biomaterial for a leukocyte adsorption removal filter to be brought into contact with blood, the reaction between leukocytes and lectin is to maintain the form present in the blood without separating leukocytes, and at the operating temperature of the leukocyte adsorption removal filter. It is desirable to do.
[0014]
A sugar chain-containing substance for cell substitution is selected based on the cell surface sugar specificity characterized by lectin. As a sugar chain-containing substance, various commercially available sugar chain research reagents in which a sugar chain is incorporated into a synthetic polymer can be used, and monosaccharides are derived from Seikagaku Corporation (Japan) into vinyl monomers. There are commercially available products that have been polymerized as a result, and products that have sugars incorporated into synthetic polyacrylamide. Also, depending on the target sugar chain, substances whose sugar chain structure shows the characteristics of the molecule, such as natural polysaccharides such as chitooligosaccharides and mannan, orosomucoid which is a kind of serum protein, ovalbumin and ovomucoid in chicken egg white, etc. It can be used as a chain-containing material.
[0015]
The interaction between the biomaterial candidate material and the biomaterial screening probe referred to in the present invention is, for example, both adsorptive or non-adsorbent, and depends on the form of use of the biomaterial.
[0016]
The screening method referred to in the present invention is a method for selecting an optimal biomaterial present at an interface that comes into contact with a biological substance such as a cell.If the biomaterial candidate material is fixed on the outermost surface, a flat plate or a flat plate may be used. The material may be a flat film or particles, and more preferably, is coated on a base material whose adsorption surface area is easily controlled to be constant between the materials to be compared. The interaction with the biomaterial screening probe selected in the present invention can be measured by any of the direct and indirect methods. For example, the change in the concentration of the probe before and after the adsorption reaction with the material is quantitatively screened by UV measurement or ELISA, or the interaction between the material and the probe is directly performed by labeling the probe with a fluorescent dye or isotope in advance. This can be done by measuring.
[0017]
The material screened by the method of the present invention is used as a biomaterial as it is or after processing such as molding into various shapes.
[0018]
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
【Example】
In this embodiment, a screening method including four steps for a biomaterial for a leukocyte adsorption removal filter will be described.
1. Determination of cells to be measured for interaction with biomaterials Since leukocyte adsorption removal filters for transfusion products interact with peripheral blood, leukocytes, erythrocytes, and platelets in blood were determined as cells to be measured for interaction. .
[0019]
2. Selection of lectins FITC-labeled Concanavalin A (ConA) and Korean Asagaorectin (DSA) purchased from Seikagaku Corporation (Japan) as lectins that recognize sugar chains on the surface of each cell membrane of leukocytes, erythrocytes, and platelets , Dog endogen lectin (MAM), peanut lectin (PNA), juvenile lectin (RCA120), gorse lectin (UEA-I), and wheat germ lectin (WGA).
[0020]
3. Selection of probe for biomaterial screening Blood was collected from healthy individuals using CPD (citrate-phosphate-dextros) as an anticoagulant. Phosphate buffer (KH ₂ PO ₄ 0.144 g / l, NaCl 9.00 g / l, Na ₂ HPO ₄ 0.795 g / l (pH 7.4)) (GIBCO BRL, Life Technologies, Inc.) 100 μl of blood diluted 1/10 in a test tube, and 0.13 to 0.20 mg / ml of various FITC-labeled lectins are added in 20 μl each, and protected from light for 30 minutes at room temperature. The blood cells and the lectin were allowed to react by standing still. A negative control was prepared by adding 20 μl of the same buffer instead of lectin and performing the same operation as described below. Each reaction was terminated by adding 1 ml of the same buffer to each reaction test tube.
Affinities of various lectins of erythrocytes, platelets, and leukocytes were measured with a flow cytometer. The flow cytometer used was FACSCalibur (BD, USA), and the data acquisition and analysis software used was CELLQuest (BD, USA). First, red blood cells, platelets, and white blood cells were gated by the forward and side scattered light detection systems, and the fluorescence intensity of FITC labeled with lectin was compared for each gate.
[0021]
FIG. 1 compares the affinity of each blood cell with each of the seven lectins. When the lectin affinity was compared between leukocytes and erythrocytes or between leukocytes and platelets, a great difference was observed between the blood cells with respect to the affinity with MAM.
Since MAM shows sugar specificity to Siaα2-3Gal, it has this sugar chain structure, and a synthetic polymer containing FITC in the molecule, Neu5Acα2-3Galβ1-4GlcNAcβ-PAA-flu (hereinafter abbreviated as 236FP) .) (Synthesome, Germany) were selected as probes for biomaterial screening.
[0022]
4. Biomaterial screening As a biomaterial candidate polymer to be screened, polyhydroxyethyl methacrylate or 2-hydroxypropyl methacrylate, dimethylamino methacrylate, methyl methacrylate, a copolymer having a molecular weight of 300,000 to 500,000 synthesized using various combinations of composition ratios is used. Was. Each polymer was dissolved in ethanol heated to 40 ° C. to prepare a 2% by weight polymer solution. Approximately 50 mg of polystyrene particles (Polybead ^™ Polystyrene Microspheres, PolyScience, USA) having a diameter of 10 μm were dispersed in 1 ml of the polymer solution to prepare a suspension having a particle concentration of 1% by weight. The particle suspension was irradiated with ultrasonic waves at 40 ° C. for 5 minutes. Thereafter, the container containing the particle suspension was attached to a rotator (small rotary incubator RT-5, TAITEC, Japan) and heated to 40 ° C. in a thermostat while rotating at a speed of 3.4 m / min. Warm and react for 55 minutes. After the reaction, the particles were separated by centrifugation, and the obtained particles were washed with distilled water and lyophilized to obtain particles coated with each polymer. The surface of each particle is surfaced with an X-ray photoelectron spectrometer (ESCA 5400, Perkin Elmer, Inc., USA) and a time-of-flight secondary ion mass spectrometer (TRIFT II, Physical Electronics, Inc., USA). By observing the molecular distribution state, it was confirmed that the entire surface of each particle was covered with the polymer.
[0023]
5 mg of each polymer-coated particle was weighed and suspended in 300 μl of a phosphate buffer containing 1% goat serum (Cappel, USA). 5 μl of the biomaterial screening probe 236FP 0.2 mg / ml selected in the step 3 was added. The adsorption of 236FP on the polymer particles was measured using a flow cytometer. In the measurement by the flow cytometer, first, a single existing particle was gated by a forward scattered light detection system, and the fraction was set to be 10,000. The 236FP adsorption rate to each polymer particle was obtained by subtracting the same measured value of each polymer particle without 236FP added as a negative control from the average value of FITC fluorescence intensity of 10,000 particles, and 236FP adsorption rate = Log (FITC fluorescence intensity− Negative control), and from these results, a polymer having a high affinity of 236FP was selected as a biomaterial for a leukocyte adsorption removal filter from among various polymers.
[0024]
In the biomaterial screening method for the purpose of developing a leukocyte adsorption removal filter, a conventional method for measuring the leukocyte adsorption removal ratio is shown below for comparison with the present invention, and FIG. 2 is a correlation diagram of data of the present invention and the conventional method. Is shown.
[0025]
Preparation of biomaterial candidate polymer-coated nonwoven fabric 10 g of each polymer was dissolved in 190 g of ethanol to prepare a 5 wt% coating solution. The made of polyethylene terephthalate fibers having an average fiber diameter of 1.2 [mu] m to coat solutions nonwoven (40 g / ^{m 2} basis weight, thickness 0.20 mm, width 0.99 mm) was allowed to continuously immersed, sandwiched nip rolls Excess coating solution was removed by passing through. The coated nonwoven fabric was dried overnight at room temperature in a drying room equipped with an exhaust duct, and then collected.
[0026]
Measurement of Leukocyte Adsorption Removal Rate Each polymer-coated nonwoven fabric was arbitrarily cut into a circular shape having a diameter of 20 mm, and 32 were filled in a filter holder (filling density: 0.2 g / cm ³ ). 15 ml of the above-mentioned healthy human blood was flowed through this column at a constant flow rate of 0.74 ml / min using a syringe pump, and 13.3 ml of the blood was collected.
[0027]
A certain amount of blood was collected from the blood before filtration and the collected liquid after filtration, and the leukocyte concentration was measured using a residual leukocyte measurement reagent system LeucoCOUNT ^™ KIT (BD Bioscience, USA). The leukocyte adsorption removal rate was calculated as -Log (the concentration of leukocytes in the collected liquid after filtration / the concentration of leukocytes in the blood before filtration).
[0028]
The adsorption rate of 236FP of the biomaterial screening probe selected from the sugar specificity of MAM characterizing the leukocyte surface and the biomaterial candidate polymer has a correlation with the leukocyte adsorption removal rate of the same polymer as shown in FIG. (Correlation coefficient 0.8123).
[0029]
【The invention's effect】
As described above, according to the present invention, biomaterials can be screened without actually using blood or the like, which saves time and cost, and eliminates the risk of infection. It is extremely important to contribute to the design and development of medical equipment.
[0030]
[Brief description of the drawings]
FIG. 1 is a diagram comparing blood cell affinity of various lectins of Example 1.
The horizontal axis indicates the type of lectin, and the vertical axis indicates the average value of the fluorescence intensity of FITC labeling the lectin of 10,000 polymer particles.
FIG. 2 is a diagram showing the correlation between the 236FP adsorption rate of Example 1 and the leukocyte adsorption removal rate of a conventional method.
The horizontal axis represents the leukocyte adsorption rate of the polymer-coated nonwoven fabric, and the vertical axis represents the 236FP adsorption rate to the polymer-coated particles.

Claims (2)

A biomaterial screening method comprising the following steps.
(1) Determine the cells whose interaction with the biomaterial is to be measured.
(2) Select a lectin that specifically binds to the cell.
(3) Select a biomaterial screening probe containing a sugar chain having affinity for the lectin.
(4) Interaction between the biomaterial screening probe and the candidate material is measured, and the biomaterial is screened from the candidate material based on the measurement result. A biomaterial that has been screened using the method according to claim 1.

Images