CN114134099A - Balanced salt solution of blood cells of marine invertebrates - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
Abstract
The invention discloses a balanced salt solution of blood cells of a marine invertebrate, which is prepared by the following steps: 1) dissolving 10-15mM KCl, 400-500mM NaCl and 20-23mM Na in each liter of deionized water2HPO410-13mM HEPES, 700U/ml penicillin 500-; 2) adjusting the pH of the solution obtained in the previous step to 7.0-7.5, and filtering and sterilizing the solution by using a 0.22 mu m filter membrane to obtain the balanced salt solution. The method is suitable for most of the blood cells of the marine invertebrates, has small damage degree to the blood cells and higher cell activity, and can protect the blood cells of the marine invertebrates from being cracked when being stored in an isotonic solution.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of marine invertebrate cell biology, in particular to a balanced salt solution of marine invertebrate blood cells.
[ background of the invention ]
The balanced salt solution is a salt solution with the electrolyte content similar to the content in blood plasma. The balanced salt solution mainly comprises inorganic salt and glucose, and has the functions of maintaining the osmotic pressure balance of cells, keeping the pH stable and providing simple nutrition. The simplest Balanced Salt Solution (BSS) is Ringer, which is a solution prepared by adding potassium chloride and calcium chloride, also called Ringer's solution, to physiological saline. Because it is invented by England physiologist "Linge", so called Linge's liquor, it is actually a commonly called compound sodium chloride injection. Ringer's Solution is also called compound sodium chloride injection because Ringer's Solution contains sodium ions, potassium ions, calcium ions, magnesium ions, chloride ions and lactate ions in addition to sodium chloride. The ringer's solution has complete components compared with normal saline, and can be used to replace normal saline to regulate body fluid, electrolyte and acid-base balance. Isotonic saline containing Na+And Cl-154mmol/L and Na in blood+And Cl-The contents of the two solutions are respectively 142mmol/L and 103mmol/L, compared with Cl of isotonic saline water-Cl content in blood-The content is high at 50mmol/L, and in severe water shortage or shock state, the kidney blood is reduced, the normal chlorine discharge function is influenced, and a large amount of isotonic saline is infused into the vein, which causes blood Cl-Too high, causing the risk of hyperchlorhydric acidosis. Thus, treatment of water deficit with isotonic saline has disadvantages, while the use of balanced saline avoids the need to introduce excessive Cl-The treatment of water deficiency is more physiological and can help to correct acidosis. Common balanced salt solutions include sodium lactate and compound sodium chloride solution (1/6M sodium lactate solution 1/3 plus compound sodium chloride solution 2/3); with both sodium bicarbonate and isotonic saline solution (1.25% sodium bicarbonate solution 1/3 and isotonic saline 2/3).
Invertebrates are generally open circulatory systems and their osmotic pressure is not known and is generally considered to be similar to the osmotic pressure of the aqueous environment in which they are located. However, in the cell culture of the marine invertebrates, the effect of the balanced salt solution prepared according to the seawater for the cell culture is not ideal, so that a proper isotonic solution formula for the marine invertebrates needs to be screened.
In order to better meet the living requirements of organisms or preserve organs, tissues or cells of the organisms, researchers invented a variety of balanced salt solutions or salt buffers, such as physiological saline, PBS buffer, artificial seawater, etc., to simulate the preservation state of the organisms in the natural environment. In a laboratory environment, the method can be properly adjusted according to specific experiments so as to meet the basic survival requirements of organisms or maintain the normal state of the organisms for more than 1 day without generating obvious harm to the organisms. At present, most balanced salt solutions including artificial seawater cannot meet the requirements of preservation and subculture of marine invertebrate cells, particularly the preservation and subculture of blood cells. Therefore, the development of a balanced salt solution formula special for the blood cells of the marine invertebrate is needed; the formula one is to ensure that these blood cells are preserved in an isotonic solution; secondly, the blood cells can be ensured to be in a survival state for a long time, and the activity of the cells is higher; thirdly, the formula is ensured not to contain calcium and magnesium ions, and the method can be widely applied to the fields of single cell transcriptome sequencing and the like.
[ summary of the invention ]
Aiming at the problem that most of the conventional salt buffer solution or balanced salt solution including artificial seawater cannot meet the requirements of preservation and subculture of marine invertebrate cells, particularly the preservation and subculture of blood cells, the invention provides the balanced salt solution of the marine invertebrate blood cells, which is suitable for most of the marine invertebrate blood cells, has small damage degree to the blood cells and higher cell activity, and can protect the marine invertebrate blood cells from being cracked when being preserved in an isotonic solution.
The purpose of the invention is realized by the following technical scheme:
a balanced salt solution of marine invertebrate blood cells prepared by the steps of:
1) dissolving 10-15mM KCl, 400-500mM NaCl and 20-23mM Na in each liter of deionized water2HPO410-13mM HEPES, penicillin 500-700U/ml, streptomycin 400600 mug/ul, nystatin 2-4 mug/ml;
2) adjusting the pH of the solution obtained in the previous step to 7.0-7.5, and filtering and sterilizing the solution by using a 0.22 mu m filter membrane to obtain the balanced salt solution;
the marine invertebrate comprises pinctada fucata, penguin shellfish, oyster, scallop, abalone, clam, prawn and mud crab.
In the invention:
in step 1), if the death rate of blood cells in the balanced salt solution is high, 0.05-0.5% (w/w) BSA solution can be additionally added into the solution.
In the step 1), 800U/ml of penicillin 400-.
Step 1) -step 2) are prepared in situ to prepare the balanced salt solution.
Compared with the prior art, the invention has the following advantages:
the balanced salt solution of the blood cells of the marine invertebrate can meet the following requirements:
1. ensuring that the blood cells of the marine invertebrate are preserved in an isotonic solution;
2. ensuring that the blood cells can be in a survival state for a long time and the activity of the cells is higher;
3. the balanced salt solution does not contain calcium and magnesium ions, and can be widely used in the fields of single-cell transcriptome sequencing and the like.
[ detailed description ] embodiments
The present invention is further described in detail with reference to the following specific examples, which are not intended to limit the invention in any manner. In addition, after reading the present disclosure, one skilled in the art can make various changes or modifications to the present disclosure, and such equivalents also fall within the scope of the present disclosure as defined by the appended claims.
Example 1:
a balanced salt solution of marine invertebrate blood cells prepared by the steps of:
1) dissolving 12mM KCl, 450mM NaCl and 20mM Na in each liter of deionized water2HPO410mM HEPES, penicillin 500U/ml, streptomycin 450 mu g/ul and nystatin 2 mu g/ml;
2) adjusting the pH value of the solution obtained in the previous step to 7.5, and filtering and sterilizing the solution by using a 0.22 mu m filter membrane to obtain the balanced salt solution;
the marine invertebrate is selected from pinctada fucata; step 1) -step 2) are prepared in situ to prepare the balanced salt solution.
The experimental process comprises the following steps:
2mL of blood was drawn from the adductor muscle of Pinctada martensii using a 1mL syringe, and pre-cooled 3-fold volume of PBS buffer and artificial seawater (26.726g/L NaCl, 2.260g/L Mgcl) were added immediately2，3.248g/L MgS04，1.153g/L CaCl2，NaHC03 0.198g/L，0.743g/L KCl，0.051g/L NaBr，0.053g/L H3B03，0.0024g/L Na2SiO3，0.0015g/L Na2Si4O9，0.002g/L H3PO4，0.018g/L Al2Cl6，0.002g/L NH3，0.0013g/L LiN03The solution is deionized water), and the pinctada fucata martensii blood cell balanced salt solution is uniformly mixed; filtering with 40 μm cell sieve, centrifuging at 4 deg.C and 1500rpm for 8min, resuspending blood cells with three times volume of the above solution, and repeating for 2 times; discarding the supernatant, adding 200 μ L of the pre-cooled solution into the precipitate to resuspend the blood cells; and (3) respectively sucking a proper amount of blood cell suspension at six time points of 0.5h, 1h, 3h, 6h, 12h and 24h, staining the blood cell suspension by using 0.04% trypan blue staining solution, and observing the cell concentration and activity by using a cell counting plate under a microscope.
The experimental results are as follows:
as shown in Table 1, the survival rate of blood cells in the Pinctada martensii blood cell balanced salt solution group is significantly greater than that of the PBS group and the artificial seawater group, and the experimental requirements of subsequent blood cell typing and function research can be met.
Table 1 example 1 comparison of blood cell viability data for each group
Example 2:
a balanced salt solution of marine invertebrate blood cells prepared by the steps of:
1) dissolving 10mM KCl, 500mM NaCl and 23mM Na in deionized water per liter2HPO413mM HEPES, 0.05% (w/w) BSA, 550U/ml penicillin, 600. mu.g/ul streptomycin and 4. mu.g/ml nystatin;
2) adjusting the pH value of the solution obtained in the previous step to 7.0, and filtering and sterilizing the solution by using a 0.22 mu m filter membrane to obtain the balanced salt solution;
the marine invertebrate is selected from abalone; step 1) -step 2) are prepared in situ to prepare the balanced salt solution.
The experimental process comprises the following steps:
2mL of blood was collected from the adductor muscle of abalone using a 1mL syringe, and the procedure was the same as in example 1;
the experimental results are as follows: as shown in table 2, the blood cell survival rate of the abalone blood cell balanced salt solution group was significantly greater than that of the PBS group and the artificial seawater group.
Table 2 example 2 comparison of blood cell viability data for each group
Example 3:
a balanced salt solution of marine invertebrate blood cells prepared by the steps of:
1) dissolving 15mM KCl, 400mM NaCl and 20mM Na in each liter of deionized water2HPO412mM HEPES, 0.1% (w/w) BSA, 600U/ml penicillin, 400. mu.g/ul streptomycin and 3. mu.g/ml nystatin;
2) adjusting the pH value of the solution obtained in the previous step to 7.5, and filtering and sterilizing the solution by using a 0.22 mu m filter membrane to obtain the balanced salt solution;
the marine invertebrate is selected from clams; step 1) -step 2) are prepared in situ to prepare the balanced salt solution.
The experimental process comprises the following steps:
2mL of blood was extracted from the clam adductor muscle using a 1mL syringe, and the procedure was the same as in example 1;
the experimental results are as follows:
as shown in table 3, the blood cell survival rate of the clam blood cell balanced salt solution group was significantly greater than that of the PBS group and the artificial seawater group.
Table 3 example 3 comparison of blood cell viability data for each group
Example 4:
a balanced salt solution of marine invertebrate blood cells prepared by the steps of:
1) dissolving 10mM KCl, 450mM NaCl and 20mM Na in deionized water per liter2HPO413mM HEPES, 0.15% (w/w) BSA, penicillin 700U/ml, streptomycin 550. mu.g/ul, nystatin 3. mu.g/ml;
2) adjusting the pH value of the solution obtained in the previous step to 7.5, and filtering and sterilizing the solution by using a 0.22 mu m filter membrane to obtain the balanced salt solution;
the marine invertebrate is selected from penaeus vannamei; step 1) -step 2) are prepared in situ to prepare the balanced salt solution.
The experimental process comprises the following steps:
2mL of blood was collected from the abdomen of Penaeus vannamei Boone using a 1mL syringe, and the procedure was the same as in example 1;
the experimental results are as follows:
as shown in table 4, the survival rate of blood cells in the penaeus vannamei boone blood cell balanced salt solution group was significantly greater than that in the PBS group and the artificial seawater group.
Table 4 example 4 comparison of blood cell viability data for each group
And (4) conclusion:
as can be seen from the above examples 1-4, the balanced salt solution of the blood cells of the marine invertebrate is suitable for most of the blood cells of the marine invertebrate, and has less damage to the blood cells and higher cell activity compared with other solutions (PBS buffer solution and artificial seawater); can meet the follow-up research on the typing and the function of the blood cells of the marine invertebrate; because the formula does not contain calcium and magnesium ions, the invention can also be widely used in the fields of marine invertebrate blood cell transcriptome sequencing, proteome sequencing, single cell transcriptome sequencing and the like.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (4)
1. A balanced salt solution of marine invertebrate blood cells characterized by: is prepared by the following steps:
1) dissolving 10-15mM KCl, 400-500mM NaCl and 20-23mM Na in each liter of deionized water2HPO410-13mM HEPES, 700U/ml penicillin 500-;
2) adjusting the pH of the solution obtained in the previous step to 7.0-7.5, and filtering and sterilizing the solution by using a 0.22 mu m filter membrane to obtain the balanced salt solution.
2. The balanced salt solution of marine invertebrate blood cells of claim 1 further comprising: the marine invertebrate comprises pinctada fucata, penguin shellfish, oyster, scallop, abalone, clam, prawn and mud crab.
3. The balanced salt solution of marine invertebrate blood cells of claim 1 further comprising: in step 1), 0.05-0.5% w/w BSA solution is additionally added to the solution.
4. The balanced salt solution of marine invertebrate blood cells of claim 1 further comprising: step 1) -step 2) are prepared in situ to prepare the balanced salt solution.
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| CN114032280A (en) * | 2021-11-29 | 2022-02-11 | 北部湾大学 | Preparation method of marine invertebrate blood cell suspension |
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