CN114134099B - Balanced salt solution for blood cells of marine invertebrate - Google Patents

Balanced salt solution for blood cells of marine invertebrate Download PDF

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CN114134099B
CN114134099B CN202111434255.5A CN202111434255A CN114134099B CN 114134099 B CN114134099 B CN 114134099B CN 202111434255 A CN202111434255 A CN 202111434255A CN 114134099 B CN114134099 B CN 114134099B
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喻达辉
王培�
白丽蓉
李素萍
郑玉斯
何积翠
郭颖
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Beibu Gulf University
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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
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract

The invention disclosesA balanced salt solution of blood cells of marine invertebrate is prepared by the following steps: 1) Dissolving 10-15mM KCl, 400-500mM NaCl and 20-23mM Na in each liter of deionized water 2 HPO 4 10-13mM HEPES, penicillin 500-700U/ml, streptomycin 400-600 μg/ul, nystatin 2-4 μg/ml; 2) The pH value of the solution obtained in the step is regulated to 7.0-7.5, and the solution is filtered and sterilized by a 0.22 mu m filter membrane to obtain the balanced salt solution. Is suitable for most of blood cells of marine invertebrates, has small damage degree to the blood cells and high cell activity, and can protect the blood cells of the marine invertebrates from being cracked when being stored in an isotonic solution.

Description

Balanced salt solution for blood cells of marine invertebrate
[ field of technology ]
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 Art ]
The balanced salt solution is a salt solution with the electrolyte content similar to the plasma content in the solution. The balanced salt solution mainly consists of 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, balanced salt solution) is Ringer, which is a solution of potassium chloride and calcium chloride, also known as Ringer's solution, added to physiological saline. Since it is invented by British physiology "ringer", it is called ringer's solution, in fact, a so-called compound sodium chloride injection. Ringer's Solution is also called compound sodium chloride injection because Ringer's Solution contains sodium, potassium, calcium, magnesium, chloride and lactate ions in addition to sodium chloride. The ringer's solution has a complete composition than normal saline solution, and can be used in place of normal saline solution to regulate body fluid, electrolyte and acid-base balance, and sodium lactate ringer is suitable for acidosis or dehydration cases with acidosis tendency, so that the operating room often causesIs used. Isotonic saline containing Na + And Cl - 154mmol/L, and Na in blood + And Cl - The contents are 142mmol/L and 103mmol/L respectively, compared with the Cl of isotonic saline - Content ratio of Cl in blood - The content is higher than 50mmol/L, and the reduction of kidney blood in severe ischemia or shock state can affect normal chlorine removal function, and a large amount of isotonic saline is infused from vein to cause blood Cl - Too high, causing the risk of high-chlorine acidosis. Thus, the use of isotonic saline to treat water deficiency has the disadvantage that the use of balanced salt solutions avoids the importation of excess 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 and compound sodium chloride solution 2/3); with sodium bicarbonate and isotonic saline solution (1.25% sodium bicarbonate solution 1/3 and isotonic saline 2/3).
Invertebrates are generally open circulatory systems, the required osmotic pressure of which is not known, and which are generally considered similar to the osmotic pressure of the aqueous environment in which they are located. However, in cell culture of sea invertebrates, the effect of a balanced salt solution prepared from sea water for cell culture is not ideal, and thus there is a need for screening suitable isotonic solution formulations for sea invertebrates.
In order to better meet the living requirements of the organism or preserve the organs, tissues or cells of the organism, researchers have 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 organism in natural environment. In the laboratory environment, the biological agent can be properly adjusted according to specific experiments so as to meet the basic living requirement of organisms or maintain the normal state of the organisms for more than 1 day, and can be used without obvious harm to the organisms. Most balanced salt solutions at present comprise artificial seawater, which cannot meet the requirements of preservation and subculture of marine invertebrate cells, in particular blood cells. Therefore, there is a need to develop a balanced salt solution formulation specific to blood cells of marine invertebrates; the formulation ensures that the blood cells are preserved in an isotonic solution; secondly, ensuring that the blood cells can be in a survival state for a long time, and the cell activity is high; thirdly, the formula does not contain calcium and magnesium ions, and can be widely used in the fields of single cell transcriptome sequencing and the like.
[ invention ]
Aiming at the problems that most of salt buffers or balanced salt solutions including artificial seawater cannot meet the requirements of preservation and subculture of marine invertebrate cells, particularly 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 preserved in an isotonic solution and not being cracked.
The aim of the invention is achieved by the following technical scheme:
a balanced salt solution of blood cells of marine invertebrate is prepared by the following steps:
1) Dissolving 10-15mM KCl, 400-500mM NaCl and 20-23mM Na in each liter of deionized water 2 HPO 4 10-13mM HEPES, penicillin 500-700U/ml, streptomycin 400-600 μg/ul, nystatin 2-4 μg/ml;
2) Regulating the pH value of the solution obtained in the step to 7.0-7.5, and filtering and sterilizing by using a 0.22 mu m filter membrane to obtain the balanced salt solution;
the marine invertebrate comprises Pinctada martensii, penguin, oyster, scallop, abalone, clam, prawn and blue crab.
In the invention, the following components are added:
in step 1), if the mortality of blood cells in the balanced salt solution is high, 0.05-0.5% (w/w) BSA solution may be additionally added to the solution.
In step 1), penicillin 400-800U/ml, streptomycin 400-600. Mu.g/ul, and nystatin 1-4. Mu.g/ml are added to prevent bacterial or fungal contamination of the balanced salt solution during preparation.
Steps 1) -2) are now ready 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 remain in the isotonic solution;
2. ensuring that the blood cells can be in a survival state for a long time and have higher cell activity;
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 ] of the invention
The present invention will be described in further detail with reference to the following specific examples, which are not intended to limit the invention in any way. Further, after reading the description of the invention, a person skilled in the art may make various modifications or adaptations to the invention, these equivalents also falling within the scope of the invention as defined in the claims appended hereto.
Example 1:
a balanced salt solution of blood cells of marine invertebrate is prepared by the following steps:
1) Dissolving 12mM KCl, 450mM NaCl and 20mM Na in each liter of deionized water 2 HPO 4 10mM HEPES, penicillin 500U/ml, streptomycin 450. Mu.g/ul, nystatin 2. Mu.g/ml;
2) Adjusting the pH value of the solution obtained in the 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 martensii; steps 1) -2) are now ready to prepare the balanced salt solution.
The experimental process comprises the following steps:
blood 2mL was withdrawn from the adductor muscle of Pinctada martensii by a 1mL syringe, and pre-chilled 3 times volume PBS buffer and artificial seawater (26.726 g/L NaCl,2.260g/L Mgcl) were immediately added, respectively 2 ，3.248g/L MgS0 4 ，1.153g/L CaCl 2 ，NaHC0 3 0.198g/L，0.743g/L KCl，0.051g/L NaBr，0.053g/L H 3 B0 3 ，0.0024g/L Na 2 SiO 3 ，0.0015g/L Na 2 Si 4 O 9 ，0.002g/L H 3 PO 4 ，0.018g/L Al 2 Cl 6 ，0.002g/L NH 3 ，0.0013g/L LiN0 3 The solution is deionized water), and the pinctada martensii blood cell balancing salt solution are uniformly mixed; filtering with 40 μm cell sieve, centrifuging at 4deg.C and 1500rpm for 8min, and re-suspending blood cells with three times of the above solution, and repeating the above steps for 2 times; discarding the supernatant, adding 200 mu L of the precooled solution to the sediment to resuspend blood cells; appropriate amounts of blood cell suspensions were aspirated at six time points of 0.5h, 1h, 3h, 6h, 12h, 24h, stained with 0.04% trypan blue dye, and observed for cell concentration and activity under a microscope with a cell counting plate.
Experimental results:
as shown in Table 1, the survival rate of blood cells in the pinctada martensii blood cell balanced salt solution group is obviously higher than that in the PBS group and the artificial seawater group, and the experimental requirements of the follow-up blood cell typing and function research can be met.
Table 1 comparison of the blood cell viability data of example 1 groups
Figure GDA0004221559720000041
Example 2:
a balanced salt solution of blood cells of marine invertebrate is prepared by the following steps:
1) 10mM KCl, 500mM NaCl, 23mM Na were dissolved in each liter of deionized water 2 HPO 4 13mM HEPES, 0.05% (w/w) BSA, penicillin 550U/ml, streptomycin 600. Mu.g/ul, nystatin 4. Mu.g/ml;
2) Adjusting the pH value of the solution obtained in the 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; steps 1) -2) are now ready to prepare the balanced salt solution.
The experimental process comprises the following steps:
blood was drawn at the adductor muscle of abalone with a 1mL syringe for 2mL, the remainder of the procedure being the same as in example 1;
experimental results: as shown in table 2, the blood cell viability 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 comparison of the blood cell viability data of example 2 groups
Figure GDA0004221559720000042
Example 3:
a balanced salt solution of blood cells of marine invertebrate is prepared by the following steps:
1) Dissolving 15mM KCl, 400mM NaCl and 20mM Na in each liter of deionized water 2 HPO 4 12mM HEPES, 0.1% (w/w) BSA, penicillin 600U/ml, streptomycin 400. Mu.g/ul, nystatin 3. Mu.g/ml;
2) Adjusting the pH value of the solution obtained in the 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; steps 1) -2) are now ready to prepare the balanced salt solution.
The experimental process comprises the following steps:
blood was drawn at the clam's adductor muscle using a 1mL syringe for 2mL, the remainder of the procedure being the same as in example 1;
experimental results:
as shown in table 3, the blood cell viability 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 comparison of the blood cell viability data of example 3 groups
Figure GDA0004221559720000051
Example 4:
a balanced salt solution of blood cells of marine invertebrate is prepared by the following steps:
1) Per literDissolving 10mM KCl, 450mM NaCl and 20mM Na in deionized water 2 HPO 4 13mM 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 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 boone; steps 1) -2) are now ready to prepare the balanced salt solution.
The experimental process comprises the following steps:
2mL of blood was drawn from the abdomen of Penaeus vannamei Boone using a 1mL syringe, and the rest of the procedure was the same as in example 1;
experimental results:
as shown in table 4, the survival rate of the blood cells of the penaeus vannamei blood cell balance salt solution group was significantly higher than that of the PBS group and the artificial seawater group.
Table 4 comparison of the blood cell viability data of example 4 groups
Figure GDA0004221559720000061
Conclusion:
it can be seen from the above examples 1-4 that the balanced salt solution of the blood cells of the marine invertebrate according to the present invention is suitable for most blood cells of the marine invertebrate, and has less damage to blood cells and higher cell activity than other solutions (PBS buffer solution, artificial seawater); can meet the follow-up research on the typing and the functions of blood cells of marine invertebrates; because the formula does not contain calcium and magnesium ions, the invention can also be widely applied to the fields of sequencing of blood cell transcriptome, sequencing of proteome, sequencing of single cell transcriptome and the like of marine invertebrate.
The foregoing description is directed to the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the invention, and all equivalent changes or modifications made under the technical spirit of the present invention should be construed to fall within the scope of the present invention.

Claims (2)

1. A balanced salt solution of blood cells of a marine invertebrate, characterized by: the preparation method comprises the following steps:
1) Dissolving 10-15mM KCl, 400-500mM NaCl and 20-23mM Na in each liter of deionized water 2 HPO 4 10-13mM HEPES, penicillin 500-700U/ml, streptomycin 400-600 μg/ul, nystatin 2-4 μg/ml;
2) The pH value of the solution obtained in the step is regulated to 7.0-7.5, and the solution is filtered and sterilized by a 0.22 mu m filter membrane to obtain the balanced salt solution.
2. A balanced salt solution of blood cells of a marine invertebrate as claimed in claim 1, characterized in that: in step 1), 0.05-0.5% w/w BSA solution is additionally added to the solution.
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