CN113117172A - Blood simulation medium and preparation method and application thereof - Google Patents
Blood simulation medium and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/14—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis
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- Health & Medical Sciences (AREA)
- Heart & Thoracic Surgery (AREA)
- Anesthesiology (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Engineering & Computer Science (AREA)
- Emergency Medicine (AREA)
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- Life Sciences & Earth Sciences (AREA)
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a blood simulation medium, which consists of a solid part and a liquid part, wherein the solid part is a fluorescent cross-linked blending microsphere used for simulating blood cells, the fluorescent cross-linked blending microsphere is prepared by starch, fluorescein and a cross-linking agent through an inverse suspension method, the liquid is a solution prepared from glycerol, polyvinyl alcohol and water and used as simulated plasma, and the two are mixed according to a certain proportion to obtain the blood simulation medium. The fluid medium with similar rheological parameters to blood can be obtained by controlling the mass ratio of the simulated blood cells to the simulated plasma. The medium can replace blood to be used for testing the structural performance in the blood perfusion device, can test the flow resistance of the blood, the pressure at two ends of a product, the exhaust function and the like, and the fluorescent microspheres can observe the movement direction of the blood in the product, the existence of a liquid flow channel and the like through an optical instrument so as to facilitate the structural design or the optimization and improvement of the blood perfusion product. The blood simulation medium has stable property, can be repeatedly used, and can be preserved for a long time after being sterilized.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a blood simulation medium and a preparation method and application thereof.
Background
Blood perfusion is a blood purification technique in which blood of a patient is introduced into a blood perfusion device filled with an adsorbent, and exogenous or endogenous toxins, drugs or metabolic waste in the blood are removed by adsorption. The blood perfusion is mainly used for rescuing drugs and toxication, and can also be used together with hemodialysis to remove macromolecular toxins in the body of a chronic renal failure maintenance dialysis patient. The structure of the hemoperfusion apparatus mainly comprises an adsorbent, a shell cylinder, a net rack with a filter screen, an end cover and an end cap. The shape, size and internal structure layout of the perfusion device influence the clinical treatment effect of the perfusion device, and even can cause clinical problems such as blood coagulation, hemolysis and air embolism in the using process of patients. If the column of the hemoperfusion device is designed to be too narrow, the blood resistance is too large, and hemolysis is caused when the tolerance degree of red blood cells is exceeded; if the inner structure layout is not reasonable, air bubbles are difficult to be expelled during treatment and enter the body of a patient along with blood, and air embolism can be caused. Therefore, the measurement of the data of the blood resistance, the pressure difference between two ends of the column, the suspension motion state of the adsorbent and the like of the perfusion device in use is very important, and the suitable test medium is the premise of scientific and accurate measurement. For hemoperfusion products, the adoption of human blood as a test medium is undoubtedly consistent with practical application, the measured result can best reflect the real state, but the blood is expensive and resource-deficient, and cannot be recycled, so that the blood is not the most suitable test medium, and therefore, a low-cost, easily-available and suitable simulation medium is urgently needed to replace the blood as the test medium.
At present, although animal blood and artificial plasma are examples of test media for measurement, there are various problems. Like animal blood and human blood, the price is expensive, and the blood is not easy to store and can not be recycled; similarly, the use of cells to simulate the formation of blood still faces the problems of high cost, difficult storage and recycling. The artificial plasma or plasma substitute, which is mainly a colloidal solution close to plasma albumin, is not suitable for being used as a test medium for the inner structure of the blood perfusion apparatus because the solution does not contain visible components such as blood cells and belongs to Newtonian fluid. Blood is a non-Newtonian fluid, and the shear stress and the shear strain rate of the non-Newtonian fluid are not linear, so that the result measured by using the artificial plasma is quite different from the actual application, and the actual condition of the product cannot be reflected. At present, although there are studies on artificial blood, the artificial blood is mainly used for simulating the function of red blood cells to realize the research of oxygen carrying and oxygen transferring to the body, and is not used as a fluid medium for evaluating the internal structure. Thus, there is currently no suitable test medium for a hemoperfusion product or an extracorporeal circulation medical device.
Disclosure of Invention
In order to solve the technical problems of the testing medium of the current blood perfusion product, the invention provides a blood simulation medium and a preparation method and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows: a blood simulation medium is provided, which consists of a solid simulating blood cells and a liquid simulating plasma, wherein the solid is a fluorescent cross-linked blending microsphere with the particle size of 2-30 mu m, the liquid is a solution with the viscosity of 1.2-20.0 mPa.s, and the mass ratio of the solid to the liquid is (0.4-1.0): 1.
In some specific embodiments, the composition of the liquid includes glycerin, polyvinyl alcohol, and water.
In some specific embodiments, the polyvinyl alcohol has an alcoholysis level of 98% and an average molecular weight of 10 to 20 ten thousand.
In some specific embodiments, the raw material of the fluorescent cross-linked blended microsphere comprises starch, fluorescein and a cross-linking agent, and the mass ratio of the fluorescein to the starch is (0.001-0.02): 1.
In some specific embodiments, the fluorescein is any one of 5-aminofluorescein, 6-aminofluorescein, and 6- (N-6-hydroxyhexyl) amide fluorescein.
In some specific embodiments, the crosslinking agent is any one of epichlorohydrin, a homolog of epichlorohydrin, 1, 4-butanediol diglycidyl ether, and a homolog of 1, 4-butanediol diglycidyl ether.
The invention also provides a preparation method of the blood simulation medium, which comprises the following steps:
synthesizing fluorescent cross-linked blending microspheres: adding starch and fluorescein into 2-3mol/L alkaline solvent, stirring uniformly, gelatinizing for 20-60min, and cooling to room temperature to obtain gelatinized blend; placing the oil phase solvent and the crosslinking agent in a container, adding the emulsifier, ultrasonically stirring for 5-10min, uniformly mixing, and preheating in 45-55 deg.C constant temperature water bath for 10-15min to obtain a preheated substance; adding the gelatinized blend into the preheated material, stirring at the speed of 800-1000rpm, and stirring and reacting at the constant temperature of 45-55 ℃ for 6-8h to prepare a mixture; washing the mixture with ethyl acetate, absolute ethyl alcohol and purified water for 8-10 times, and separating out 2-30 μm fluorescent cross-linked mixed microspheres;
preparation of a simulated plasma solution: dissolving polyvinyl alcohol in hot water of 90-100 ℃, cooling to room temperature, and adding glycerol and water to prepare a simulated plasma solution;
preparing a blood simulation medium: and mixing the fluorescent cross-linked blending microspheres and the simulated plasma solution according to a proportion to obtain the blood simulated medium.
In some specific embodiments, the oil phase solvent is liquid paraffin or petroleum ether.
In some specific embodiments, the emulsifier is a span-based or Tween-based emulsifier.
The invention also provides an application of the blood simulation medium, and the blood simulation medium is used as a test medium to be applied to the test of the hemoperfusion apparatus.
The technical scheme provided by the embodiment of the application at least has the following technical effects or advantages:
the blood simulation medium consists of a solid and a liquid, wherein the solid is a fluorescent cross-linked blending microsphere used for simulating blood cells, the liquid is a solution with certain viscosity used for simulating blood plasma, and the solid and the liquid are mixed according to a certain proportion to obtain the blood simulation medium. The fluid medium with similar rheological parameters to blood can be obtained by controlling the mass ratio of the fluorescence crosslinking blending microspheres and the simulated plasma solution. The medium can replace blood to be used for testing the structural performance in the blood perfusion device, can test the flow resistance of the blood, the pressure at two ends of a product, the exhaust function and the like, and the fluorescent microspheres can observe the movement direction of the blood in the product, the existence of a liquid flow channel and the like through an optical instrument so as to facilitate the structural design or the optimization and improvement of the blood perfusion product. The blood simulation medium has stable property, can be repeatedly used, and can be preserved for a long time after being sterilized.
Drawings
FIG. 1 is a comparison graph of the arteriovenous pressure difference between hemoperfusion A and B (artificial plasma);
FIG. 2 is a comparison graph of the difference between the arterial and venous pressures of hemoperfusion A and B (human blood);
FIG. 3 is a comparison graph of the arteriovenous pressure difference of hemoperfusion A and B (simulated medium).
Detailed Description
The present invention is described in detail below by way of examples to facilitate understanding of the present invention by those skilled in the art, and it is to be specifically noted that the examples are provided only for the purpose of further illustrating the present invention and are not to be construed as limiting the scope of the present invention.
The invention relates to a blood simulation medium, which consists of a solid part and a liquid part, wherein: the solid is a fluorescent cross-linked blending microsphere with the particle size of 2-30 mu m and is used for simulating blood cells; the liquid is a solution with certain viscosity and is used for simulating blood plasma, and the mass ratio of the solid to the liquid is (0.4-1.0): 1.
As a preferred embodiment, the raw materials of the fluorescent cross-linked blending microsphere comprise starch, fluorescein and a cross-linking agent, wherein the mass ratio of the fluorescein to the starch is (0.001-0.02): 1.
The starch is polysaccharide polymer compound, can contain straight chain and branched chain, and can be corn starch, cassava starch, etc.
The fluorescein may be any one of 5-aminofluorescein, 6-aminofluorescein and 6- (N-6-hydroxyhexyl) amide fluorescein, and is preferably 5-aminofluorescein.
The cross-linking agent is any one of epichlorohydrin, epichlorohydrin homologues, 1, 4-butanediol diglycidyl ether and 1, 4-butanediol diglycidyl ether homologues, and is mainly used for improving the solubility resistance, temperature resistance, dimensional stability, mechanical strength and the like of the microspheres.
As a preferred example, the composition of the simulated plasma includes glycerol, polyvinyl alcohol and water. The simulated plasma comprises the following components in percentage by weight: 5-30% of glycerin, 0.1-1% of polyvinyl alcohol and the balance of water. The alcoholysis degree of the polyvinyl alcohol is 98 percent, and the average molecular weight is 10-20 ten thousand.
The application also provides a preparation method of the blood simulation medium, which comprises the following steps:
synthesizing fluorescent cross-linked blending microspheres: adding starch and fluorescein into 2-3mol/L alkaline solvent, stirring uniformly, gelatinizing for 20-60min, wherein the alkaline solvent is preferably NaOH aqueous solution, and cooling to room temperature to obtain gelatinized blend; placing the cross-linked suspending agent in a container, adding emulsifier, ultrasonically stirring for 5-10min, uniformly mixing, and preheating in 45-55 deg.C constant temperature water bath for 10-15min to obtain preheated substance; adding the gelatinized blend into a preheating material, stirring at the speed of 800-1000rpm, and stirring and reacting at the constant temperature of 45-55 ℃ for 6-8h to prepare a mixture; and (3) pouring the mixture, sequentially adopting ethyl acetate, absolute ethyl alcohol and purified water to respectively wash for 8-10 times, and preparing the fluorescent cross-linked blending microsphere. Wherein: ethyl acetate is used as an organic solvent for cleaning organic residues such as oil phase/emulsifier, cross-linking agent, unreacted fluorescein and the like; absolute ethanol is also used to clean oil phase/emulsifier, cross-linking agent, unreacted fluorescein and other organic residues, and also used to clean ethyl acetate; the purified water is used for cleaning the residue of the emulsifier, the absolute ethyl alcohol and the alkaline solvent. The fluorescent cross-linked blending microspheres with a certain particle size range are obtained by screening through a screening pot, and are soaked in purified water for storage for later use, wherein the spheres of the fluorescent cross-linked blending microspheres can be prevented from being dehydrated and collapsed by soaking in the purified water.
Preparation of a simulated plasma solution: dissolving polyvinyl alcohol in hot water of 90-100 ℃, cooling to room temperature, and adding glycerol and water to prepare a simulated plasma solution;
preparing a blood simulation medium: and mixing the fluorescent cross-linked blending microspheres and the simulated plasma solution according to the proportion to obtain the blood simulated medium.
The fluorescent cross-linked blending microsphere is prepared by dissolving starch and fluorescein as matrixes in an alkaline solvent for blending, cross-linking with a cross-linking agent and carrying out a reverse suspension balling method. The starch and fluorescein blended solution is dispersed in organic solution (oil phase) containing cross-linking agent and emulsifier to form uniform and stable water-in-oil (W/O) type inverse emulsion, then the starch high molecular chains are cross-linked into balls under high-speed stirring, and the mixed microspheres are formed by cross-linking agent cross-linking, so that the performance of the mixed microspheres is stable, the mixed microspheres can be repeatedly used, and the mixed microspheres can be stored for a long time after being sterilized. The fluorescence of the starch microspheres can be obtained by adding the fluorescein, and the fluid can be qualitatively observed more intuitively or quantitatively tested by an optical instrument in the test fluid.
The oil phase of the reversed phase suspension method is preferably liquid paraffin or petroleum ether, and as the oil phase of the reversed phase suspension, the emulsifier of the reaction system can be span type or Tween type, and is preferably any one or more of span20, span60, Tween20, Tween60 and Tween 80.
The application also provides an application of the blood simulation medium, the blood simulation medium is applied to the test of the blood perfusion device as a test medium, the blood simulation medium can replace blood to be used for testing the structural performance in the blood perfusion device, the flow resistance of the blood, the pressure at two ends of a product, the exhaust function and the like can be tested, the microsphere with fluorescence can observe the movement direction of the blood in the product through an optical instrument, whether a liquid flow channel exists or not and the like, so that the structural design or the optimization improvement of the blood perfusion product are facilitated.
Example 1
Preparation of mock blood cells: 150g of starch and 1.0g of 5-aminofluorescein are weighed, added into 500ml of 2mol/L NaOH aqueous solution, stirred uniformly and gelatinized for 30min, and cooled to room temperature to form a blend. Putting 150ml of epoxy chloropropane and 1500ml of liquid paraffin into a three-neck flask, adding 50g of emulsifier span 80, and stirring and mixing uniformly; and (3) putting the uniformly mixed three-neck flask into a constant-temperature water bath kettle at 50 ℃ for preheating for 10min, adding the gelatinized blend into the three-neck flask, stirring at 800rpm, and stirring at 50 ℃ for reacting for 8 h. And after the reaction is finished, pouring the mixture in the three-neck flask into a sand core funnel, washing the mixture for 10 times by using ethyl acetate, washing the mixture for 10 times by using absolute ethyl alcohol and washing the mixture for 10 times by using purified water, and screening the cleaned mixture by using a screen pot with a filter screen to obtain the fluorescent cross-linked blended microspheres, wherein the particle size distribution range of the fluorescent cross-linked blended microspheres is 5-25 mu m.
Preparation of mock plasma: accurately weighing 2.0g of polyvinyl alcohol (PVA-124) to dissolve in 100ml of hot water, cooling to room temperature, adding 250g of glycerol and 700g of water into a beaker, and uniformly stirring to obtain a simulated plasma solution, wherein the viscosity is 7.04 +/-0.92 mPa & s when the shear rate is 50/s.
Preparation of blood-simulating medium: weighing 400g of fluorescent cross-linked blend microspheres and 700ml of simulated plasma solution, and mixing to obtain the simulated plasma, wherein the simulated plasma can observe fluorescence under the irradiation of an ultraviolet lamp.
Example 2
Preparation of mock blood cells: 150g of starch and 1.5g of 6-aminofluorescein are weighed, added into 500ml of 2mol/L NaOH aqueous solution, stirred uniformly and gelatinized for 30min, and cooled to room temperature to form a blend. Putting 200ml of epoxy chloropropane and 1500ml of liquid paraffin into a three-neck flask, adding 60g of emulsifier span 80, and stirring and mixing uniformly; and (3) placing the uniformly mixed three-neck flask into a constant-temperature water bath kettle at 50 ℃ for preheating for 10min, adding the gelatinized blend into the three-neck flask, stirring at 1000rpm, and stirring at 50 ℃ for reacting for 8 h. And after the reaction is finished, pouring the mixture in the flask into a sand core funnel, washing the mixture for 10 times by using ethyl acetate, washing the mixture for 10 times by using absolute ethyl alcohol and washing the mixture for 10 times by using purified water, and screening the cleaned mixture by using a screen pot with a filter screen to obtain the fluorescent cross-linked blending microspheres, wherein the particle size distribution range of the fluorescent cross-linked blending microspheres is 10-30 microns.
Preparation of mock plasma: accurately weighing 2.5g polyvinyl alcohol (PVA-124) and dissolving in 100ml hot water, cooling to room temperature, adding 300g glycerol and 800g water into a beaker, and stirring to obtain simulated plasma solution with shear rate of 50/s and viscosity of 9.28 + -0.78 mPa.s.
Preparation of blood-simulating medium: weighing 300g of fluorescent cross-linked blend microspheres and 700ml of simulated plasma solution, and mixing to obtain the simulated plasma, wherein the simulated plasma can observe fluorescence under the irradiation of an ultraviolet lamp.
Example 3
The blood simulation medium prepared by the methods of the embodiment 1 and the embodiment 2 and the blood of the healthy person are respectively measured by a rotary viscometer, and the results show that the viscosity of the blood simulation medium prepared by the methods can be configured into the range of the normal blood viscosity of the person (the viscosity under high, medium and low shear rates) and also can be configured into high viscosity. The viscosity changes along with the change of the shear rate, and the non-Newtonian fluid conforms to the property of the non-Newtonian fluid. The results are shown in Table 1 below:
TABLE 1 shear Rate vs. viscosity for examples 1 and 2 and human blood
Example 4
The blood simulative medium prepared in example 1 is used as a test medium to test the blood perfusion apparatuses A and B of two different manufacturers on the market, and the two perfusion apparatuses have similar shape specifications but slightly different internal structures. An extracorporeal circulation loop is formed by the hemoperfusion apparatus, the blood pump, the blood pipeline and the like in a simulated clinical use mode, the pressure gauge monitors the arteriovenous pressure of the hemoperfusion apparatus, and human blood and artificial plasma (4.76mPa & s) are used as a contrast test, and the test results are shown in figures 1-3. As can be seen from the figure: the change rule of the arteriovenous pressure of the prepared simulated medium along with the flow velocity is consistent with that of human blood, so that the difference of the arteriovenous pressure difference of different perfusion devices can be reflected, and the difference of the arteriovenous pressure difference of two perfusion devices cannot be detected by artificial blood plasma.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are intended to be within the scope of the invention.
Claims (10)
1. A blood-mimicking medium, comprising: the medium consists of a solid simulating blood cells and a liquid simulating plasma, wherein the solid is a fluorescent cross-linked blended microsphere with the particle size of 2-30 mu m, the liquid is a solution with the viscosity of 1.2-20.0mPa & s, and the mass ratio of the solid to the liquid is (0.4-1.0): 1.
2. A blood simulation medium according to claim 1, wherein: the composition of the liquid includes glycerin, polyvinyl alcohol, and water.
3. A blood simulation medium according to claim 2, wherein: the alcoholysis degree of the polyvinyl alcohol is 98%, and the average molecular weight of the polyvinyl alcohol is 10-20 ten thousand.
4. A blood simulation medium according to claim 1, wherein: the raw materials of the fluorescent cross-linking blending microsphere comprise starch, fluorescein and a cross-linking agent, wherein the mass ratio of the fluorescein to the starch is (0.001-0.02): 1.
5. A blood simulation medium according to claim 4, wherein: the fluorescein is any one of 5-aminofluorescein, 6-aminofluorescein and 6- (N-6-hydroxyhexyl) amide fluorescein.
6. A blood simulation medium according to claim 4, wherein: the cross-linking agent is any one of epichlorohydrin, homologues of epichlorohydrin, 1, 4-butanediol diglycidyl ether and homologues of 1, 4-butanediol diglycidyl ether.
7. A method of preparing a blood mimicking medium according to any one of claims 1-6, comprising the steps of:
synthesizing fluorescent cross-linked blending microspheres: adding starch and fluorescein into 2-3mol/L alkaline solvent, stirring uniformly, gelatinizing for 20-60min, and cooling to room temperature to obtain gelatinized blend; placing the oil phase solvent and the crosslinking agent in a container, adding the emulsifier, ultrasonically stirring for 5-10min, uniformly mixing, and preheating in 45-55 deg.C constant temperature water bath for 10-15min to obtain a preheated substance; adding the gelatinized blend into the preheated material, stirring at the speed of 800-1000rpm, and stirring and reacting at the constant temperature of 45-55 ℃ for 6-8h to prepare a mixture; washing the mixture with ethyl acetate, absolute ethyl alcohol and purified water for 8-10 times, and separating out 2-30 μm fluorescent cross-linked mixed microspheres;
preparation of a simulated plasma solution: dissolving polyvinyl alcohol in hot water of 90-100 ℃, cooling to room temperature, and adding glycerol and water to prepare a simulated plasma solution;
preparing a blood simulation medium: and mixing the fluorescent cross-linked blending microspheres and the simulated plasma solution according to a proportion to obtain the blood simulated medium.
8. A blood simulation medium according to claim 7, wherein: the oil phase solvent is liquid paraffin or petroleum ether.
9. A blood simulation medium according to claim 7, wherein: the emulsifier is span emulsifier or Tween emulsifier.
10. Use of a blood simulant medium according to any one of claims 1 to 6 as a test medium in the testing of a hemoperfusion cartridge.
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