CN101919381B - Preserving fluid of hepatic cells for biological artificial liver and preparation method thereof - Google Patents
Preserving fluid of hepatic cells for biological artificial liver and preparation method thereof Download PDFInfo
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Abstract
The invention provides preserving fluid of hepatic cells for a biological artificial liver and a preparation method thereof. The preserving fluid is a solution compounded by ultrapure water. The solution contains the following components within the concentration range: 15-25mmol/L of disodium hydrogen phosphate, 1-10mmol/L of sodium hydrogen phosphate dehydrate, 4-6mmol/L of potassium citrate monohydrate, 10-30mmol/L of sodium chloride, 5-10mmol/L of magnesium chloride hexahydrate, 3-10mmol/L of disodium adenosine triphosphate, 1-5mmol/L of reducing glutathione, 0.1-0.5mmol/L of alpha-lipoic acid, 100-150mmol/L of trehalose (C6H12O5), 200/0.510-50g/L of hydroxyethyl starch and 2-10mg/L of matrine. The preparation method of the preserving fluid comprises the following steps of: accurately weighing all components according to the concentration requirements of the components, wherein the alpha-lipoic acid is weighed in a dark place; completely dissolving the other components except the alpha-lipoic acid by using the right amount of ultrapure water; sufficiently dissolving the alpha-lipoic acid in the dark place; and adding the ultrapure water to full dose. The preserving fluid can well protect the cell activity of the hepatic cells for the biological artificial liver and the special functions of the hepatic cells at low temperature so as to satisfy the short-term low temperature preservation of a large-scale hepatic cell bank for the biological artificial liver and/or the hepatic cell protection in the long-distance transportation process.
Description
Technical field
The present invention relates to the external preservation liquid biological technical field of organ, tissue or the cell of human body or animal, especially relate to preservation liquid of a kind of hepatic cells for biological artificial liver and preparation method thereof.
Background technology
China is the district occurred frequently of hepatopathy, annual approximately 1,200,000 examples of acute viral hepatitis that occur, hepatitis gravis account for wherein 0.2%～0.4%, case fatality rate can be up to 70%.Approximately have 1% can develop into acute or subacute liver failure in hepatitis B patient.Approximately have 20% acute hepatitis b patient to be converted into chronic, approximately 3% patient can develop into posthepatitic cirrhosis.Approximately 60%～80% be converted into chronicly in the third hepatopath, 20% can develop into liver cirrhosis, and 12% develops into hepatopathy in whole latter stage.Die from approximately 300,000 examples of liver patient every year, wherein 50% is primary hepatocellular carcinoma, mostly relevant with second, hepatitis C.Therefore hepatitis gravis, liver failure and hepatitis hepatopathy liver cirrhosis in whole latter stage and liver cancer etc. are China liver problem sufferer main causes of death.Liver transplantation is the most thorough and effective means of this type of disease for the treatment of.The liver transplantation of China is counted every year more than 700 examples, and survival rate surpassed 50% in 1 year, and the live time that lives forever most was over 6 years.But how China's severe viral hepatitis lacks for organ, and many patients can not get timely during orthotopic liver transplantation and lose chance of surviving, and effectively the donor resource also fails to take full advantage of simultaneously.Therefore the new treatment of the cell levelss such as bioartificial liver and hepatocyte transplantation has obtained research and development.Hepatocyte transplantation and bioartificial liver are supported in a large amount of experimentation on animalies and some clinical experiments and obtain good result, become promising liver disease approach.In fact, if hepatocyte transplantation and bioartificial liver in clinical a large amount of utilizations, hepatocellular demand will increase, the situation of the donor deficiency of equally facing with organ transplantation also will occur.If a practical low-temperature storage technology is reliably arranged, set up a liver cell storehouse, critical patient can obtain at any time a large amount of high motility rates, the liver cell of function is arranged; Hepatocyte transplantation and bioartificial liver's technology just can generally be promoted.
In biological artificial liver support system, most important parts are exactly bio-reactor.Yet team that has wide experience of the synthetic needs of bio-reactor carries out the amplification of cell with specific installation under aseptic condition and the stdn of function detects, and this just needs a center---" cell factory " goes to provide bio-reactor to load the amplification of cell and is transported to the hospital that needs.Porcine hepatocyte loads cell as bio-reactor and all obviously descends through Cryopreserved-viability rate and the peculiar function of liver cell, has greatly limited the result for the treatment of of artificial liver.Therefore, after liver cell amplification is completed, the short period of time preserve and the long-distance transportation process in need that a kind of convenience is arranged, effective, practical protection loads the method for cell viability and function.4 ℃ is organ and mass cell group's standard storage temperature, and transportation need not can reach requirement by specific installation at this temperature.Hepatocellular injury is mainly that low temperature injury and rewarming damage again in 4 ℃ of preservation processes, is similar to the ischemical reperfusion injury in the organ transplantation process.Belzer etc. are according to the various pathologic, physiologic characteristics of organ under low temperature; a kind of composition of effective organ hypothermia protection liquid has been proposed; the characteristics that must possess following five aspects: 1. reduce the acidification that 2. edema that causes because of low temperature prevent cell and prevent that 3. 4. the swelling in the intercellular substance in the lavation process from preventing the damage of oxyradical, especially 5. provide the substrate of regeneration high-energy phosphate compound 6. to keep cell homeostasis in refilling process.
Main Organ Preservation Solution mainly contains the UW liquid of U.S.'s design and the HTK liquid of European design in the market.UW liquid is to use that the most a kind of many organ perfusions preserve liquid in present world wide; can effectively preserve kidney 72h, pancreas 72h, liver 30h, heart 24h; but it still has many deficiencies, and its hypertonicity causes that cell shrinkage, high viscosity affect the medicine, expensive that situ perfusion, high potassium do not meet physiology, lack protection liver cryopreservation.HTK liquid is the same with UW liquid is comparatively ideal organ preservative fluid, the advantage of HTK liquid maximum is its low viscosity, can improve the microcirculation of preserving organ, improve tissue oxidizing etc., it still has obvious deficiency to be mainly reflected in the restriction of organ shelf time, along with the non-functional incidence of prolongation graft of time can significantly promote.
Summary of the invention
The technical problem to be solved in the present invention is to overcome defects; preservation liquid of a kind of hepatic cells for biological artificial liver and preparation method thereof is provided; this preserve liquid can be under low-temperature condition cell viability and the peculiar function of liver cell of fine protection hepatic cells for biological artificial liver, with satisfy that the bioartificial liver reaches with the short-term cryopreservation in extensive liver cell storehouse or the long distance transportation process in hepatocellular protection.
The invention provides a kind of preservation liquid of hepatic cells for biological artificial liver, be the solution of ultrapure water preparation, comprise the component of following concentration range in this solution:
The PH of this solution is 7.2-7.4, and osmotic pressure is 305 ± 10mmol/L.
Preferably, the component that comprises following concentration in described solution:
the preservation liquid making method of hepatic cells for biological artificial liver of the present invention is: according to above-mentioned formula with medicine electronic balance accurate weighing, dissolve other compositions except alpha-lipoic acid fully with appropriate ultrapure water, after lucifuge accurate weighing alpha-lipoic acid, fully dissolving under the lucifuge state, add ultrapure water to capacity, measure pH value under 4 ℃ of conditions, osmotic pressure, vacuum filter (0.22 μ m) filters, sample drawn is done the bacterium cultivation, with the liquid brown PVP bag of packing into, put 4 ℃ of Refrigerator stores standby, gained liquid is water white transparency, without muddy, without precipitation and floss.The aforesaid operations process is all completed in the sterile preparation chamber, and utensil used all passes through aseptically process.
The basal component that the preservation liquid of hepatic cells for biological artificial liver of the present invention contains is to keep hepatocellular vigor and ionogen physiological concentration under low-temperature condition, its contained component is mainly to alleviate acidifying in cell, alleviates oxygen free radical injury, stabilized cell membrane structure, replenishes the edema that cellular energy is lost, minimizing low temperature causes.On formulating of recipe, the advantage that has absorbed UW liquid, HTK liquid has possessed own significantly characteristics simultaneously:
1. has powerful surge capability.Choose two kinds of bufferings right, HPO
4/ H
2PO
4And two kinds of bufferings of Citrate trianion (citrate) are right, prevent that cell is in the acidification of low-temperature condition.
2. select impermeability protective material trehalose (treha lose), play the effect of stabilizing cell membrane and protein structure, alleviate edema under low-temperature condition.
3. add extracellular ATP, magnesium chloride as the substrate of regeneration high-energy phosphate compound, passed through ATP-Mg
2+For cell provides energy.
4. the alpha-lipoic acid of title that adopts reduced glutathion and universal antioxidant is arranged is as antioxidant system and anti-Apoptosis System, prevents or alleviates cell reperfusion injury, the damage of radical pair cell.
5. introduce the activeconstituents matrine of Chinese medicine, utilize its oxidation-resistance, strengthen provide protection.
6. use hydroxyethylamyle, improve the method for oncotic pressure, alleviate edema.
Description of drawings
Fig. 1 is that after preservation liquid of the present invention, UW liquid, 4 ℃ of cryopreservation cells of Ringer lactate solution difference, cell survival rate compares schematic diagram;
Fig. 2 a, 2b, 2c are preservation liquid of the present invention, UW liquid, the Ringer lactate solution two X400 microscopic examination figure that dye of cell AO/PI after 4 ℃ of cryopreservation cell 24h respectively;
Fig. 3 a, 3b, 3c are preservation liquid of the present invention, UW liquid, the Ringer lactate solution two X400 microscopic examination figure that dye of cell AO/PI after 4 ℃ of cryopreservation cell 48h respectively;
Fig. 4 a, 4b, 4c are preservation liquid of the present invention, UW liquid, the Ringer lactate solution two X400 microscopic examination figure that dye of cell AO/PI after 4 ℃ of cryopreservation cell 72h respectively;
Fig. 5 is that after preservation liquid of the present invention, UW liquid, 4 ℃ of cryopreservation cells of Ringer lactate solution difference, the cell albumin content compares schematic diagram.
Embodiment
Below the preferred embodiments of the present invention are described.
Embodiment one:
The preservation liquid I (1000ml) of preparation hepatic cells for biological artificial liver of the present invention
1, add the 800ml ultrapure water in the 1000ml container;
2, add the 7163mg disodium hydrogen phosphate, stirring is fully dissolved it;
3, add the 780mg sodium dihydrogen phosphate dihydrate, stirring is fully dissolved it;
4, add 1622mg Citric Acid Monohydrate potassium, stirring is fully dissolved it;
5, add 1168mg sodium-chlor, stirring is fully dissolved it;
6, add the 1827mg magnesium chloride hexahydrate, stirring is fully dissolved it;
7, add the 2755mg Sodium ATP, stirring is fully dissolved it;
8, add the 922mg reduced glutathion, stirring is fully dissolved it;
9, add the 47287.5mg trehalose, stirring is fully dissolved it;
10, add 30g Hetastarch 200/0.5, stirring is fully dissolved it;
11, add the 6mg matrine, stirring is fully dissolved it;
12, add the 41mg alpha-lipoic acid under the lucifuge state, and stirring is fully dissolved it;
13, capacity adjustment: add ultrapure water until overall solution volume is 1000ml, stirring makes solution be liquid even, colourless, clarification;
14, after active carbon filtration, measure pH value under 4 ℃ of conditions and be 7.3, osmotic pressure is 305mmol/L, vacuum filter (0.22 μ m) filtration sterilization, sample drawn is done the bacterium cultivation.
Embodiment two:
The preservation liquid II (1000ml) of preparation hepatic cells for biological artificial liver of the present invention, compound method is identical with embodiment one, and the consumption of component is: the 5372mg disodium hydrogen phosphate, the 1560mg sodium dihydrogen phosphate dihydrate, 1298mg Citric Acid Monohydrate potassium, 585mg sodium-chlor, 2030mg magnesium chloride hexahydrate, the 5510mg Sodium ATP, the 307mg reduced glutathion, 56745mg trehalose, 50g Hetastarch 200/0.5, the 2mg matrine, the 103mg alpha-lipoic acid; After active carbon filtration, measure pH value under 4 ℃ of conditions and be 7.2, osmotic pressure is 315mmol/L, vacuum filter (0.22 μ m) filtration sterilization, sample drawn is done the bacterium cultivation.
Embodiment three:
The preservation liquid III (1000ml) of preparation hepatic cells for biological artificial liver of the present invention, compound method is identical with embodiment one, and the consumption of component is: the 8950mg disodium hydrogen phosphate, the 156mg sodium dihydrogen phosphate dihydrate, 1946mg Citric Acid Monohydrate potassium, 1755mg sodium-chlor, 1015mg magnesium chloride hexahydrate, the 1653mg Sodium ATP, the 1535mg reduced glutathion, 37830mg trehalose, 10g Hetastarch 200/0.5, the 10mg matrine, the 20.6mg alpha-lipoic acid; After active carbon filtration, measure pH value under 4 ℃ of conditions and be 7.4, osmotic pressure is 295mmol/L, vacuum filter (0.22 μ m) filtration sterilization, sample drawn is done the bacterium cultivation.
The present invention preserves liquid at the preservation effect of hepatic cells for biological artificial liver for research, and the preservation liquid I, II, the III that prepare with above-described embodiment test, and hepatic cells for biological artificial liver C3A is experimental cell.With Lactated Ringer'S Solution, UW liquid carries out the experiment of rewarming to 37 ℃ cell survival rate, apoptosis and peculiar function thereof after preserving 24h, 48h, 72h under 4 ℃ of cold condition for contrast.
One, after cryopreservation, cell survival rate detects
Experimental technique:
1, cell cultures: digestion collector liver cell (C3A), adjusting cell density is 3 * 10
5/ ml is seeded in six well culture plates, and every hole liquid volume added reaches 2ml, and 37 ℃, 5%CO
2, carbonic acid gas incubator adherent culture is 4 ℃ of cryopreservation of row after 24 hours.
2, cryopreservation: absorb original each hole nutrient solution, PBS2ml rinses.Add respectively the following protective material of respectively organizing of 2ml, be respectively; Preserve liquid I, UW protection liquid and Ringer lactate solution, be placed in 4 ℃ of refrigerators and preserved 24,48,72 hours.
3, cell is inserted after 4 ℃ of refrigerators take out fast 37 ℃ of water-baths after water-bath recovery 2mim, the careful exhaustion refuses to dye test determination with 0.4% placenta indigo plant after each group is preserved liquid.Its survival rate of viable cell percentage test in 200 liver cells of random five visuals field counting under opticmicroscope.
Experimental result:
Embodiment one preserves liquid I group and UW liquid group human liver cell (C3A) survival rate 24h, the equal no significant difference of 48h, and after 72h, embodiment one preserves liquid I group human liver cell C3A) survival rate is a little less than UW liquid group.Negative control group Lactated Ringer'S Solution group human liver cell (C3A) survival rate is namely seen obvious decline after preserving 24h, referring to Fig. 1.
Test equally to preserve liquid II, III, come to the same thing.
Two, cell apoptosis assay after cryopreservation
Experimental technique:
1, cell cultures: digestion collector liver cell (C3A), adjusting cell density is 3 * 10
5/ ml is seeded in six well culture plates, and every hole liquid volume added reaches 2ml, and 37 ℃, 5%CO2, carbonic acid gas incubator adherent culture is 4 ℃ of cryopreservation of row after 24 hours.
2, cryopreservation: absorb original each hole nutrient solution, PBS2ml rinses.Add respectively the following protective material of respectively organizing of 2ml, be respectively; Preserve liquid I, UW protection liquid and Ringer lactate solution, be placed in 4 ℃ of refrigerators and preserved 24,48,72 hours.
3, acridine orange (AO)/two dying of iodate the third ingot (PI): fluorescent microscope detects the apoptosis situation.
Experimental result:
Dye result as seen by upper different preservation liquid group human liver cell (C3A) acridine orange/propidium iodides are two, preserving liquid I compares with UW liquid group, two dying has no obvious apoptotic cell after preserving 24h, 48h, two groups of no significant differences, and see more apoptosis after 72h, and karyon pyknosis shape, being green fluorescence enhancing, bright, effect is slightly poor (sees Fig. 2 a, Fig. 2 b, Fig. 3 a, Fig. 3 b, Fig. 4 a, Fig. 4 b).
And negative control group Lactated Ringer'S Solution group sees obviously that namely apoptosis appears in liver cell under low-temperature condition after preserving 24h, and karyon is pyknosis shape, crumb structure, is green fluorescence enhancing, bright, even presents " Huang " look.The visible necrosis of 48h, 72h or late period apoptosis, core is that larger circular configuration orange, or orange and be the pyknosis shape or round bead shape (seeing Fig. 2 c, Fig. 3 c, Fig. 4 c).
Test equally to preserve liquid II, III, come to the same thing.
Three, liver cell albumin complex functionality after cryopreservation
Experimental technique:
1, cell cultures: digestion collector liver cell (C3A), adjusting cell density is 2 * 10
6/ ml is seeded in six well culture plates, and every hole liquid volume added reaches 2ml, and 37 ℃, 5%CO2, carbonic acid gas incubator adherent culture is 4 ℃ of cryopreservation of row after 24 hours.
2, cryopreservation: absorb original each hole nutrient solution, PBS2ml rinses.Add respectively the following protective material of respectively organizing of 2ml, be respectively; Preserve liquid I, UW protection liquid and Ringer lactate solution, be placed in 4 ℃ of refrigerators and preserved 24,48,72 hours.
3, cell is inserted after 4 ℃ of refrigerators take out fast 37 ℃ of water-baths after water-bath recovery 2mi m, careful exhaust each group and preserve liquid after, add in each group and contain 10% foetal calf serum DMEM cell culture medium cellar culture 24,48,72 hours.Regularly collect the nutrient solution supernatant, detect human albumin content in culture supernatant with human albumin radioimmunological kit (northern Institute for Atomic Research provides), adopt radioimmunity antigenic competition method, with the bovine serum albumin no cross reaction.
Experimental result: albumin complex functionality there was no significant difference during preservation liquid I group and UW liquid group are recovered after cryopreservation 24h, 48h and cultivated, negative control group Lactated Ringer'S Solution group and other two experimental group have significant difference at cell recovery postalbumin complex functionality.(seeing Fig. 5)
Test equally to preserve liquid II, III, come to the same thing.
By the proof of above experiment, the present invention is significant effective ground 4 ℃ of cryopreservation bioartificial livers with human liver cell, particularly in cryopreservation 48 hours, on cell survival rate and the peculiar function of cell thereof with the UW liquor ratio than no significant difference.The present invention simultaneously preserves the contained component of liquid and originates abundant, cost is low, be different from previously cell-preservation liquid, each component of the present invention has definite composition and concentration, it mainly is comprised of disaccharide, saccharan, polypeptide, metal ion, phosphate anion etc., cause does not need to add other heterologous antigens and the cytotoxic dimethyl sulfoxide (DMSO) such as serum, the antigen-antibody rejection after the dangerous and introducing heterologous antigen of having avoided being polluted by unknown virus.Thereby cell need not namely can be used for bioartificial liver's bio-reactor through special processing after cryopreservation, fine satisfied supply that clinical bioartificial liver requires the liver cell material fast, the requirement of emergent (ready-to-use).
Above disclosed is only the preferred embodiments of the present invention, certainly can not limit with this interest field of the present invention, and the equivalent variations of therefore doing according to the present patent application the scope of the claims still belongs to the scope that the present invention is contained.
Claims (7)
1. the preservation liquid of a hepatic cells for biological artificial liver, the solution for the ultrapure water preparation is characterized in that, comprises the component of following concentration range in this solution:
2. the preservation liquid of hepatic cells for biological artificial liver as claimed in claim 1, is characterized in that, the pH of described solution is 7.2-7.4.
3. the preservation liquid of hepatic cells for biological artificial liver as claimed in claim 1, is characterized in that, the osmotic pressure of described solution is 305 ± 10mmol/L.
4. the preservation liquid of hepatic cells for biological artificial liver as claimed in claim 1, is characterized in that, comprises the component of following concentration in described solution:
5. the preparation method of the preservation liquid of hepatic cells for biological artificial liver as claimed in claim 1, is characterized in that, comprises the steps:
According to the concentration requirement of described component with the accurate weighing of described each component, alpha-lipoic acid lucifuge weighing wherein;
Dissolve other components except alpha-lipoic acid fully with appropriate ultrapure water;
Fully dissolve alpha-lipoic acid under the lucifuge state;
Add ultrapure water to capacity.
6. the preparation method of the preservation liquid of hepatic cells for biological artificial liver as claimed in claim 5, is characterized in that, also comprises the steps: to filter to the capacity step at the described ultrapure water that adds; With the brown PVP bag of packing into of the solution after filtering, put Refrigerator store standby.
7. the preparation method of the preservation liquid of hepatic cells for biological artificial liver as claimed in claim 6, is characterized in that, described step is all completed in the sterile preparation chamber, and utensil used all passes through aseptically process.
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| CN103404509B (en) * | 2013-08-07 | 2015-04-08 | 彭乐 | Cell preserving fluid as well as preparation method and application of cell preserving fluid |
| CN104054695B (en) * | 2014-07-04 | 2016-02-17 | 中国水产科学研究院黑龙江水产研究所 | Biological tissue's cryoprotective agent and preparation, using method |
| CN104819888B (en) * | 2015-03-30 | 2018-01-09 | 中国农业科学院兰州兽医研究所 | A kind of ELISA sample diluting liquids and preparation method thereof |
| CN104922059A (en) * | 2015-05-21 | 2015-09-23 | 北京青藤谷禧干细胞科技研究院有限公司 | Umbilical cord mesenchymal stem cell injection and preparation method and application thereof |
| CN105532646B (en) * | 2016-01-27 | 2017-10-13 | 武汉仝干医疗科技股份有限公司 | Liver cell preserves liquid and its preparation method and application |
| CN106614525A (en) * | 2016-12-19 | 2017-05-10 | 深圳市麦瑞科林科技有限公司 | Cell preservation solution as well as preparation method and use method thereof |
| CN111972399B (en) * | 2020-08-06 | 2021-11-30 | 温州医科大学 | Preservation solution for maintaining activity of liver cells |
| CN114586768A (en) * | 2020-12-03 | 2022-06-07 | 江苏齐氏生物科技有限公司 | Liver tissue preservation solution and preservation method and application thereof |
| CN114271262B (en) * | 2021-12-09 | 2023-05-12 | 四川大学华西医院 | Multi-organ preservation solution and application thereof |
| CN113925049B (en) * | 2021-12-17 | 2022-04-29 | 广东乾晖生物科技有限公司 | Cell preservation solution for maintaining cell activity and preparation method and application thereof |
| CN114831107B (en) * | 2022-04-11 | 2023-02-24 | 苏州依科赛生物科技股份有限公司 | Low-temperature preservation solution for cells and tissues as well as preparation method and application thereof |
| CN114958724A (en) * | 2022-06-26 | 2022-08-30 | 依雨大健康科技(广州)有限公司 | Production method of stable primary hepatocyte kit |
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