CN111172108A - Preparation method of tissue fluid - Google Patents
Preparation method of tissue fluid Download PDFInfo
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- CN111172108A CN111172108A CN201911173446.3A CN201911173446A CN111172108A CN 111172108 A CN111172108 A CN 111172108A CN 201911173446 A CN201911173446 A CN 201911173446A CN 111172108 A CN111172108 A CN 111172108A
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Abstract
The invention is suitable for the field of biomedical experiments, and provides a preparation method of tissue fluid, which comprises the following steps: step one, collecting blood of healthy people or experimental animals; step two, performing anticoagulation or non-anticoagulation treatment on the blood, and centrifuging to obtain plasma or serum; step three, adding the serum or the plasma into a filter of an ultrafiltration tube, embedding the filter into a nested tube, performing centrifugal operation, and collecting liquid in the nested tube to obtain a pretreatment liquid; regulating the pH value of the pretreatment solution to 7.1-7.3 by using a sodium citrate buffer solution to obtain a primary tissue fluid; and step five, filtering and sterilizing the preliminary tissue fluid by using a 0.22 micron microporous filter membrane to obtain the tissue fluid. The tissue fluid components obtained by the method are close to the real tissue fluid, the requirements of biomedical experiments can be met, and the safety is good.
Description
Technical Field
The invention belongs to the field of biomedical experiments, and particularly relates to a preparation method of tissue fluid.
Background
Interstitial fluid is fluid that exists between cells and is also referred to as interstitial fluid. The tissue fluid exists in animal and plant bodies in large quantity, is a liquid environment for the direct life of common cells, and can exchange substances with the cells. The cells are infiltrated in the tissue fluid and can exchange substances with the tissue fluid, which is a necessary condition for ordinary cells to metabolize. In addition, the tissue fluid can also have buffering and protecting effects on cells.
In the animal body, the tissue fluid permeates into a part of fluid in the tissue gap from the arterial end of the capillary vessel, exchanges substances with tissue cells, and then flows back to blood or lymph through the venous end of the capillary vessel or the lymphatic capillary. Most of the tissue fluid is in a gel state, can not flow freely and can not flow to the low-drop part of the body under the action of gravity; the injection needle is inserted into the tissue space and cannot extract the tissue fluid. Therefore, interstitial fluid is difficult to obtain directly under physiological conditions, and the composition thereof is not completely determined. The interstitial fluid is generated by the filtration of plasma through the wall of the blood vessel at the arterial end of the blood capillary, and most of the interstitial fluid is absorbed and recovered through the wall of the blood vessel at the venous end of the blood capillary. Under normal conditions, interstitial fluid is continuously generated from the arterial end of the capillary vessel, meanwhile, one part of the interstitial fluid returns to the capillary vessel through the capillary vein end, and the other part of the interstitial fluid returns to the blood circulation through the lymphatic vessel. Interstitial fluid is formed as plasma filters through the walls of capillaries. Thus, it is generally accepted that interstitial fluid has a composition similar to the fluid components in blood. The chemical components of the blood plasma comprise 90-92% of water, and solutes mainly comprise plasma proteins. The various chemical components of blood plasma constantly vary within a certain range, wherein the concentrations of glucose, proteins, fats, hormones and the like are most susceptible to the nutritional status and the activity of the body, and the concentration of inorganic salts varies in a small range.
The acquisition of tissue fluid is of great significance for the study of the composition of tissue fluid and for further biomedical applications. The main components of interstitial fluid and blood differ in the ratio of blood cells and plasma proteins: the tissue fluid contains few blood cells and the plasma protein content is relatively low. The major plasma protein components in plasma include: albumin with a molecular weight of 67kD at about 58%; about 38% globulin, molecular weight 150-196 kD; and a small amount of fibrinogen with a molecular weight of about 340kD, and other trace proteins such as regulatory proteins and coagulation factors. In the past, the tissue fluid simulation fluid is obtained by configuring different components, and the other tissue fluid simulation fluid is extracted from body tissues through some special devices. The former has a large difference from the real tissue fluid, while the latter has a limited volume and is expensive.
Disclosure of Invention
The invention aims to solve the problems that the existing tissue simulation fluid has large difference between the components and the real tissue fluid and the quantity of the tissue fluid extracted from the body tissue is limited, and provides a preparation method closer to the tissue fluid, and the obtained tissue fluid is closer to the real tissue fluid.
In order to solve the technical problem, the invention is realized by a method for preparing tissue fluid, which comprises the following steps:
step one, collecting blood of healthy people or experimental animals;
step two, performing anticoagulation or non-anticoagulation treatment on the blood, and centrifuging to obtain plasma or serum;
step three, adding the serum or the plasma into a filter of an ultrafiltration tube, embedding the filter into a nested tube, performing centrifugal operation, and collecting liquid in the nested tube to obtain a pretreatment liquid;
regulating the pH value of the pretreatment solution to 7.1-7.3 by using a sodium citrate buffer solution to obtain a primary tissue fluid;
and step five, filtering and sterilizing the preliminary tissue fluid by using a 0.22 micron microporous filter membrane to obtain the tissue fluid.
Compared with the prior art, the invention has the advantages of simple process, mild conditions, high content of bioactive components such as amino acid, polypeptide, lipid, saccharides and the like in the product, high activity, good safety, low production cost of the product, and components close to real tissue fluid, and can meet the requirements of biomedical experiments.
Drawings
FIG. 1 is a flow chart of the preparation of interstitial fluid according to an embodiment of the present invention.
FIG. 2 is a graph showing the concentration of proteins in plasma and serum in interstitial fluid obtained in example 1 of the present invention.
FIG. 3 is a graph showing the contents of endotoxin in plasma and serum in interstitial fluid obtained in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
To achieve the object of the present invention, an embodiment of the present invention provides a method for preparing tissue fluid, including the following steps:
step one, collecting blood of healthy people or experimental animals;
step two, performing anticoagulation or non-anticoagulation treatment on the blood, and centrifuging to obtain plasma or serum;
step three, adding the serum or the plasma into a filter of an ultrafiltration tube, embedding the filter into a nested tube, performing centrifugal operation, and collecting liquid in the nested tube to obtain a pretreatment liquid;
regulating the pH value of the pretreatment solution to 7.1-7.3 by using a sodium citrate buffer solution to obtain a primary tissue fluid;
and step five, filtering and sterilizing the preliminary tissue fluid by using a 0.22 micron microporous filter membrane to obtain the tissue fluid.
In particular, the process of collecting blood from healthy humans or experimental animals is kept sterile, avoiding contamination.
In the second step, the anticoagulated or non-anticoagulated blood is preferably left to stand at room temperature for 1.5 to 5 hours, more preferably at room temperature for 3 hours before centrifugation. The rotation speed of the centrifugation is 1500-5000 revolutions per minute, and the more preferable rotation speed is 3000 revolutions per minute; the time for centrifugation is 3 to 10 minutes, and more preferably 5 minutes.
Preferably, an anticoagulant is added to the serum, the anticoagulant is added when plasma is separated, and the anticoagulant is at least one of heparin, Ethylene Diamine Tetraacetic Acid (EDTA), sodium citrate and sodium oxalate, and is further preferably heparin or EDTA. Specifically, 0.8mg EDTA was added to 1mL of blood, and 0.1mg heparin was added to 1mL of blood.
In the third step, the molecular weight cut-off of the selected ultrafiltration tube is 5-100 kD, preferably 10-50 kD, the centrifugal speed is 10000-14000 r/min, preferably 12000 r/min, and the centrifugal time is 10-20 min, preferably 15 min.
In the fourth step, the pH value of the sodium citrate buffer solution is 4.0-6.0.
The preparation method of the tissue fluid has simple process, as shown in a preparation flow chart of the tissue fluid in figure 1, mild conditions, low production cost and components close to those of real tissue fluid, keeps most of the components such as amino acid, polypeptide, sugar, lipid and the like in the tissue fluid, can meet the requirements of biomedical experiments, and has good safety.
Specific implementations of the present invention are described in detail below with reference to specific embodiments.
Example 1
1. Collecting blood of healthy person
4mL of fresh blood of healthy people is collected by using a blood anticoagulation tube with an EDTA anticoagulant, and the blood collection process keeps aseptic operation.
2. Separation of plasma
Centrifuging the collected anticoagulation blood at 3000 rpm for 5 min, and collecting the supernatant with a pipette after centrifuging to obtain plasma.
3. Separation of plasma proteins
Plasma was added to 30kD molecular weight ultrafiltration tubes, nested into nested tubes, and centrifuged at 12000 rpm for 10 minutes at high speed. And after the centrifugation is finished, collecting the liquid in the nested tubes to obtain a primary tissue fluid.
4. Adjusting the pH value
The pH value of the preliminarily obtained tissue fluid is detected by a pH meter, and a proper amount of citrate buffer is added to the tissue fluid to adjust the pH value to 7.2.
5. Filtration sterilization
Filtering the tissue fluid by using a 0.22 micron filter, sterilizing the primary tissue fluid by using a 0.22 micron filter membrane, and collecting the filtrate to obtain a final tissue fluid product.
Example 2
1. Collecting blood of healthy person
4mL of fresh blood of healthy people is collected by using a common blood collection tube, and the blood collection process keeps aseptic operation.
2. Separation of serum
And standing the collected blood at room temperature for 3 hours, centrifuging at the rotating speed of 3000 rpm for 5 minutes, and collecting the supernatant by using a pipette after the centrifugation is finished to obtain the serum.
3. Separation of plasma proteins
Adding the serum into an ultrafiltration tube with the molecular weight of 30kD, nesting the ultrafiltration tube into a nested tube, and centrifuging at a high speed of 12000 rpm for 10 minutes. And after the centrifugation is finished, collecting the liquid in the nested tubes to obtain a primary tissue fluid.
4. Adjusting the pH value
The pH value of the preliminarily obtained tissue fluid is detected by a pH meter, and a proper amount of citrate buffer is added to the tissue fluid to adjust the pH value to 7.2.
5. Filtration sterilization
Filtering the tissue fluid by using a 0.22 micron filter, sterilizing the primary tissue fluid by using a 0.22 micron filter membrane, and collecting the filtrate to obtain a final tissue fluid product.
Example 3
1. Collecting blood of SD rat for experiment
One section of the needle of the blood anticoagulation tube with EDTA anticoagulant is inserted into the abdominal aorta of the SD rat, 4mL of fresh blood is collected, and the blood collection process keeps aseptic operation.
2. Separation of plasma
Centrifuging the collected anticoagulation blood at 3000 rpm for 5 min, and collecting the supernatant with a pipette after centrifuging to obtain plasma.
3. Separation of plasma proteins
Plasma was added to 30kD molecular weight ultrafiltration tubes, nested into nested tubes, and centrifuged at 12000 rpm for 10 minutes at high speed. And after the centrifugation is finished, collecting the liquid in the nested tubes to obtain a primary tissue fluid.
4. Adjusting the pH value
The pH value of the preliminarily obtained tissue fluid is detected by a pH meter, and a proper amount of citrate buffer is added to the tissue fluid to adjust the pH value to 7.2.
5. Filtration sterilization
Filtering the tissue fluid by using a 0.22 micron filter, sterilizing the primary tissue fluid by using a 0.22 micron filter membrane, and collecting the filtrate to obtain a final tissue fluid product.
The BCA kit is used for carrying out protein quantification on the tissue fluid collected in the example 1, and the specific operation flow is as follows: the albumin standard was serially diluted to obtain the following 9 standards with different concentrations: 2000. mu.g/mL, 1500. mu.g/mL, 1000. mu.g/mL, 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL, 31.25. mu.g/mL, 0. mu.g/mL. Taking 25 mu L of each of the albumin standard substance and the sample to be detected into a 96-well plate, adding 200 mu L of BCA working solution into each well, and covering a 96-well plate cover. The 96-well plate was left at 37 ℃ for 30 minutes, and the light absorption at 562nm was measured by a microplate reader. As shown in FIG. 2, the concentration of albumin in the tissue fluid obtained in example 1 was reduced to 5mg/mL or less, indicating that the tissue fluid obtained by this method had a composition characteristic similar to that of the real tissue fluid, in which the proteins having a large molecular weight were partially removed.
The method for detecting the endotoxin content of the tissue fluid prepared in the example 1 by using the limulus kit comprises the following operation procedures: 1. preparing endotoxin standard solutions comprising 10EU/mL, 1EU/mL, 0.1EU/mL and 0.01EU/mL, and setting water for bacterial endotoxin detection as a negative control; 2. adding water for detecting bacterial endotoxin into the chromogenic substrate according to the marked amount, slightly shaking to dissolve the chromogenic substrate, then adding all the dissolved chromogenic substrate into the limulus reagent, and slightly shaking to completely dissolve the limulus reagent; 3. taking a pyrogen-removing microplate, and respectively adding 100 mu L of water for bacterial endotoxin detection, endotoxin standard solution, interstitial fluid and plasma into each hole; 4. the microplate was incubated at 37 ℃ for 120 minutes and then the absorbance at 405nm was measured using a microplate reader. The result shows that the content of endotoxin in the tissue fluid prepared by the method is similar to that in original serum or plasma and is lower than the online requirement of biological product detection, and the tissue fluid prepared by the method meets the requirement of the biological product.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. A method for preparing tissue fluid is characterized by comprising the following steps:
step one, collecting blood of healthy people or experimental animals;
step two, performing anticoagulation or non-anticoagulation treatment on the blood, and centrifuging to obtain plasma or serum;
step three, adding the serum or the plasma into a filter of an ultrafiltration tube, embedding the filter into a nested tube, performing centrifugal operation, and collecting liquid in the nested tube to obtain a pretreatment liquid;
regulating the pH value of the pretreatment solution to 7.1-7.3 by using a sodium citrate buffer solution to obtain a primary tissue fluid;
and step five, filtering and sterilizing the preliminary tissue fluid by using a 0.22 micron microporous filter membrane to obtain the tissue fluid.
2. The method for preparing tissue fluid according to claim 1, wherein the anticoagulation agent is at least one of heparin, ethylenediaminetetraacetic acid, sodium citrate, and sodium oxalate.
3. The method for preparing tissue fluid according to claim 1 or 2, wherein the anticoagulation agent is heparin or ethylenediaminetetraacetic acid.
4. The method for preparing tissue fluid according to claim 1, wherein in the second step, the centrifugation speed is 1500-5000 rpm, and the centrifugation time is 3-10 minutes.
5. The method for preparing a tissue fluid according to claim 1, wherein in the second step, the anticoagulated or non-anticoagulated blood is allowed to stand at room temperature for 1.5 to 5 hours before centrifugation.
6. The method of claim 1, wherein the ultrafiltration tube has a molecular weight cut-off of 5 to 100 kD.
7. The method of claim 1 or 6, wherein the ultrafiltration tube has a molecular weight cut-off of 10-50 kD.
8. The method for preparing tissue fluid according to claim 1, wherein in step three, the centrifugal rotation speed is 10000-14000 rpm.
9. The method for preparing tissue fluid according to claim 1, wherein in step three, the centrifugation time is 10 to 20 minutes.
10. The method of claim 1, wherein the pH of the buffered sodium citrate solution is 4.0 to 6.0.
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| CN201911173446.3A CN111172108A (en) | 2019-11-26 | 2019-11-26 | Preparation method of tissue fluid |
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| CN201911173446.3A CN111172108A (en) | 2019-11-26 | 2019-11-26 | Preparation method of tissue fluid |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113828426A (en) * | 2021-09-07 | 2021-12-24 | 康膝生物医疗(深圳)有限公司 | Method and device for extracting platelet-rich plasma from blood |
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| US4156681A (en) * | 1974-03-28 | 1979-05-29 | Plasmesco Ag | Process for isolating albumin from blood |
| CN1569031A (en) * | 2004-04-26 | 2005-01-26 | 巫山 | Plasma component series preparation by inactivating virus and sieving sectionally by hyperfiltration membrane |
| CN102599332A (en) * | 2012-03-09 | 2012-07-25 | 上海杰隆生物制品股份有限公司 | Method for producing low-ash poultry plasma protein powder by poultry blood |
| US20130236984A1 (en) * | 2007-08-08 | 2013-09-12 | School Juridical Person Kitasato Institute | Method for concentration of low-molecular-weight proteins and peptides in body fluid sample |
| CN107136296A (en) * | 2017-06-02 | 2017-09-08 | 中国农业科学院农产品加工研究所 | High gelation SDPP and preparation method thereof |
| CN108813090A (en) * | 2018-06-06 | 2018-11-16 | 江苏省农业科学院 | The preparation method of poultry plasma protein |
-
2019
- 2019-11-26 CN CN201911173446.3A patent/CN111172108A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4156681A (en) * | 1974-03-28 | 1979-05-29 | Plasmesco Ag | Process for isolating albumin from blood |
| CN1569031A (en) * | 2004-04-26 | 2005-01-26 | 巫山 | Plasma component series preparation by inactivating virus and sieving sectionally by hyperfiltration membrane |
| US20130236984A1 (en) * | 2007-08-08 | 2013-09-12 | School Juridical Person Kitasato Institute | Method for concentration of low-molecular-weight proteins and peptides in body fluid sample |
| CN102599332A (en) * | 2012-03-09 | 2012-07-25 | 上海杰隆生物制品股份有限公司 | Method for producing low-ash poultry plasma protein powder by poultry blood |
| CN107136296A (en) * | 2017-06-02 | 2017-09-08 | 中国农业科学院农产品加工研究所 | High gelation SDPP and preparation method thereof |
| CN108813090A (en) * | 2018-06-06 | 2018-11-16 | 江苏省农业科学院 | The preparation method of poultry plasma protein |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113828426A (en) * | 2021-09-07 | 2021-12-24 | 康膝生物医疗(深圳)有限公司 | Method and device for extracting platelet-rich plasma from blood |
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