CN105886454B - A kind of formula of tissue and cell purification density gradient solution - Google Patents
A kind of formula of tissue and cell purification density gradient solution Download PDFInfo
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- CN105886454B CN105886454B CN201610205894.7A CN201610205894A CN105886454B CN 105886454 B CN105886454 B CN 105886454B CN 201610205894 A CN201610205894 A CN 201610205894A CN 105886454 B CN105886454 B CN 105886454B
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
A kind of formula of tissue and cell purification density gradient solution, it includes density gradient ingredient in being formulated, such as Iohexol, Sodium Amidotrizoate, Iodixanol, the substance for improving osmotic pressure and preventing tissue and edema is added, such as gossypose, trehalose, Magnevist Solution is added, solvent is the buffers such as culture solutions and HBSS such as DMEM.The tissue and cell density gradient separations that the present invention solves big animal and people at present are difficult, the problems such as isolated tissue and low cell activity, make those digest or mechanical dispersion after different densities cell and tissue can reach separation under centrifugal condition by this density gradient.We devise such density gradient solution, for promoting the Separation Research of tissue and cell both at home and abroad, have certain application value.The principle of the invention is simple, and preparation is easy, is easy to use, can reach large-scale production.This patent is declared as the exploitation of the density gradient separation liquid and applied to laying the foundation.
Description
Technical field
It is used cooperatively jointly the present invention relates to exclusive use during a kind of tissue and cell purification or with other solution
A kind of density gradient solution formula, and in particular to density gradient of tissue and cell purification based on the basis of cell culture fluid
Solution, the density gradient purification of tissue and cell for animal or people.
Background technique
Famous American biologist George Dai Li is said: " twentieth century is the epoch of drug therapy, and 21st century is thin
The epoch of born of the same parents' treatment ".The development that human transplant learns is one of achievement most outstanding in 20th century medicine, especially last century 70 years
Generation end is to the beginning of the eighties, and new immunosuppressor comes out, and the clinical efficacy of the survival rate and organ transplant that make graft greatly mentions
It is high.In recent years since clinical transplantation case increases significantly, the shortage of donor seems very prominent.In the 21st century, clinical transplantation
The research and application of art are again urged toward climax, cell transplantation, as bone-marrow transplantation, stem cell transplantation and islet cell transplantation are equal again
Achieve significant curative effect.
Cell transplantation refer to will refer to human organ, the tissue-derived work free in right amount with normal physiological function is thin
Born of the same parents are transplanted in the blood vessel, body cavity or histoorgan of receptor (human or animal), to reach the technology of therapeutic purposes.Cell transplantation
Clinical application is increasingly extensive, and wherein marrow attracts attention with hematopoietic stem cell transplantation, can be used for treating heredity combined immunization and lacks
Fall into the genetic diseases, Severe Aplastic Anemia such as disease, thalassemia major, and the blood including various leukaemia
The diseases such as liquid System Malignant Tumor.In addition, there are also islet cell transplantations to treat type 1 diabetes, hepatocyte transplantation treats severe liver
Scorching hepatic coma, spleen cell transplantation treat severe hemophilia A and Leydig cells transplantation treatment male sexual dysfunction
(male hypoandrogenisim) etc..
Cell transplantation (such as islet cell transplantation, bone-marrow transplantation, stem cell transplantation, hepatocyte transplantation) is in diabetes, cancer
Higher and higher application value is shown in the treatment of the diseases such as disease, hepatopathy, hereditary disease and tissue damage, in part treatment neck
Domain, as diabetes, leukaemia etc. have surmounted the treatment means of big organ transplant and chemicotherapy significantly, it has also become outer at present
The important directions of section's organ transplant development.
Cell transplantation is the new direction of surgery transplanting, is one of National 863 emphasis grant policies, belongs to emerging biology doctor
Medicine industry, research and application to cell are the new of the treatments such as cardiovascular disease, diabetes, the nervous system disease, liver diseases
Approach and new hope are the hot spots of medical domain international competition.The emerging Bio-pharmaceutical Industry have high-tech, high added value,
Advantage at low cost, pollution is few, is that government gives special assistance to direction.
Pancreatic islets transplantation is to eradicate the good method of clinical type 1 diabetes and diabetes B end late stage patients, islet cell transplantation
Because it is low to the requirement of pancreas donor, graft procedure is simple, operation, Small side effects are hardly needed, and whole pancreas transplantation is to donor
It is required that high, exocrine gland processing complexity, postoperative complications are more；In recent years with the application of novel immune Restrain measurement, pancreas islet
The basic research of cell, isolated pancreatic islet, the quantity of purifying and quality are also greatly improved, and pancreatic islets transplantation is in diabetes
Major progress is obtained in clinical treatment；According to 2012,5 years insulin did not depended on rate and has reached after pancreatic islets transplantation
50%, with whole pancreas transplantation (52%) without difference.Medical field always praises highly islet cell transplantation for treatment diabetes most
Ideal method, the clinical application of pancreatic islets transplantation have become irresistible trend, as the founder of world's transplantation of pancreas
What Sutherland doctor was foretold in world's transplanting conference, pancreatic islets transplantation is just replacing transplantation of pancreas gradually to promote in the world
Using becoming the Gospel of diabetic.In coming few decades, pancreatic islets transplantation will likely become treatment unstable 1 type of blood glucose
The optimal selection of diabetes and diabetes B end late stage patients.
Islet cell transplantation treats the research of blood glucose shakiness type type 1 diabetes and diabetes B end late stage patients complete
The world has passed by more than 40 years, and especially in 2000, the team of Canadian professor Sharpiro leader disclosed " Edmonton scheme "
After.Facts proved that pancreatic islets transplantation not only can control the blood glucose of patient, it is often more important that be able to suppress and improve complications of patients undergoing
Generation.But Edmonton pancreatic islets transplantation treatment plan is perfect not enough, patient can completely disengage insulin dependence for a long time
As a result also less desirable.The clinical application of pancreatic islets transplantation makes much progress in recent years, early in 2004, is put by U.S. NIH
1.7 hundred million dollars are carried out clinical islet separation and the standardization of transplanting program and the evaluation item of 7 new Immunotherapy regimens,
The Immunotherapy regimens of the middle entitled CIT07 of project have carried out the treatment of clinical 48 patients, and postoperative patient is all detached from insulin
Treatment, some patients completely dispensed with insulin therapy more than 4 years, and pancreas islet life span has reached whole pancreas transplantation level.Add
Put on airs, the national approved pancreatic islets transplantation in Europe and Australia etc. enters medical insurance, self pancreatic islets transplantation the U.S. also already
Into medical insurance.Now with the aggravation of aging of population, the disease incidence of domestic diabetes, pancreatic disease etc. is risen year by year,
Cell transplant techniques also will will gradually flourish at home.
The purpose of isolated pancreatic islet purifying is that complete pancreas islet is extracted from the postdigestive tissue of pancreas, the main original of the step
Reason is that islet cells than outer secretion/tracheal tissue has a lower density.If these cell types are in pancreas digestion process
In be efficiently separated, then islet cells can be purified by we by many means.In the large scale purification of people's pancreas islet
In it is most important progress be using 2991 separator cell of COBE be used for pancreas islet purifying.The centrifuge is initially used exclusively for
Cell component different in blood is separated, but the density contrast based on different tissues after pancreas digestion, the machine are also applied for separating
People's pancreas islet.
Density gradient centrifugation (density gradient centrifugation) is also known as density gradient equilibrium centrifugation.
With centrifuge to small-molecule substance solution, for a long time plus a centrifugal force field reaches sedimentation equilibrium, in settling tank from liquid level to
There is certain density gradient in bottom.If a small amount of macromolecular or tissue, cell solution are added in the solution, solution internal ratio is molten
The big part of agent density just generates sedimentation, and the part smaller than solvent density will float, finally in centrifugal force and buoyant equilibrium
The ribbon to form sediment is gathered in position.Using this phenomenon, the swim density or root of nucleic acid or protein etc. can be measured
A kind of sedimentation equilibrium method of separation cell or tissue is carried out according to its difference.From meter Xi Erxun (M.Meselson) in 1958, this tower
You are (F.W.Stahl), and since Wei Nuoge ladd (J.Vinograd) has had successfully been isolated (15N) DNA and (14N) DNA, which is taken
Obtain many achievements.This method mostly uses dense cesium chloride solution, so also using this name of Cesium chloride concentrations gradient centrifugation sometimes
Claim, the solution such as rubidium chloride, cesium bromide also can be used.
But separation for the purpose of cell or tissue etc. is to purify also is often used centrifuge and utilizes sucrose well prepared in advance
The density contrast of isodensity gradient liquid is separated.Its working principle is that: there are when sedimentation coefficient difference between variable grain, certain
Under centrifugal force effect, particle is respectively settled with certain speed, and the method for zone is formed in density gradient different zones.Density level bands
Degree medium should be pre-formed, and the maximal density of medium is less than the density of all samples particle.
For density gradient if it is discontinuous, group is woven in the density interface overlapping of similar density；Density gradient is also possible to
Continuous density gradient, this like cell can be efficiently separated according to the accurate density of gradient liquid.
There are some relevant document introductions in preparation for density gradient solution, foreign countries, and it is pure that some of them have been used for pancreas islet
Change, including sucrose, ficoll (Ficoll), dialysis ficoll (dialysed Ficoll), ficoll cardiografin (Ficoll
Hypaque), metrizamide (Metrizamide), colloidal silicon (Percoll), ficoll Kang Rui (Ficoll Conray), Portugal are poly-
Sugar (Dextran), bovine serum albumin solution, ficoll Sodium Amidotrizoate (Ficoll Diatrizoic), Iodixanol
(Odixanol) and Iohexol (Nycodenz), etc..In addition there are also the introductions to 50 Isopyknic separation density gradient medias
Say the fact that will not change pancreas islet density and the overlapping of outer secretory tissue density.It is usually used in two density level bands of people's isolated pancreatic islet purifying
Spending medium is Euro-Ficoll and hyperosmosis bovine serum albumin(BSA) (500mOsm/kg/H2O).Ficoll and bovine serum albumin
The white pros and cons for cell separation had had discussion on some documents.The advantage of Ficoll is its solution clear, can
High pressure sterilization and non-animal.Its shortcomings that is to be seeped when aggravating the aggregation and high concentration of cell by dramatically increasing in conjunction with water
Permeability energy.The advantages of bovine serum albumin(BSA) is that have certain protective effect to cell and reduce cell aggregation, the disadvantage is that deriving from
Animal is contaminated with easy.The development of isolated pancreatic islet purifying also has shown that the solution composition specific density ladder of dissolution density gradient ingredient
It is more important to spend ingredient, the fact that in the separation of other cells it has also been found that for many years.
The density gradient of isolated pancreatic islet is also constantly being improved.Such as by increasing additional hydroxyethyl starch in UW liquid
Solution or colloidal silicon (Percoll) prepare density gradient；By Ficoll density gradient (1.100g/ml) and two kinds of UW liquid
The new density gradient of solution composition, although this modify the range for reducing density gradient, it, which allows to load, more disappears
Change tissue (originally more than twice), and can be effectively separated in a wheel purifies.This increase whole pancreas to handle the time
(gradient of continuous density technology originally generally requires the purifying of 2 wheels).However, this adjustment may be not appropriate for all pancreas islet point
From center because the optimal use temperature of UW liquid is at 4 DEG C, in order to guarantee islet viability, COBE 2991 must reequip and
It is run at a temperature of this, is placed in a cold room.
In order to increase tissue or intercellular density contrast to be separated, preventing the oedema of tissue or cell is one important
Measure.This respect, which can be used, is added the impermeable osmotic pressure promotor of some macromoleculars to realize, such as gossypose, trehalose, cream
Saccharic acid potassium, hydroxyethyl starch etc..
In order to preferably keep activity of the substance to be separated in density gradient separation liquid, the present invention uses cell culture fluid
The solvent of DMEM, RPMI 1640, CMRL 1066, M199 or buffer HBSS, Hanks liquid as the density gradient separation liquid,
The experiment proved that isolated tissue and cell activity is good.
Magnevist Solution (Gadopentetate dimeglumine) is also known as Gd DTPA Glu, diethylenetriamine five
The double meglumines of gadolinium acetate, Gadopentetate Dimeglumine or magnevist, are a kind of contrast agent of common nuclear magnetic resonance.After this product intravenously administrable
It permeates into the extracellular fluid respectively organized in vivo, is then discharged through glomerular filtration with original shape quickly, have a small amount of secretion in stomach and intestine
It is discharged behind road with excrement, adverse reaction is few.Its molecular weight is 938, clinical concentration are as follows: 37%-47%.The experiment proved that will
Magnevist Solution be added to it is above-mentioned by ficoll 400, Iodixanol, Iohexol isodensity gradient components and gossypose, trehalose,
The ingredients such as lactobionic acid potassium, hydroxyethyl starch, with cell culture fluid DMEM, RPMI 1640, CMRL 1066, M199 or buffer
HBSS, Hanks solution are that can dramatically increase the separating effect of ingredient to be separated in the density gradient that solvent is made into.
The invention patent is with Iodixanol, Iohexol, Sodium Amidotrizoate, metrizamide, colloidal silicon, ficoll 400 are a kind of or
The mixing of several ingredients as main density gradient material, with gossypose, trehalose, lactobionic acid potassium, hydroxyethyl starch it is a kind of or
Several ingredients mixing as the substance for preventing tissue and edema, add Magnevist Solution, with DMEM, RPMI 1640,
One of CMRL 1066, M199 culture solution or one of its improved solution are solvent, a kind of tissue prepared
With cell density gradient separations solution.
Ficoll 400 is removed, the density gradient ingredient in the formula of density gradient solution is Iodixanol, Iohexol, general
The mixing of shadow acid sodium, colloidal silicon, metrizamide, colloidal silicon one or more ingredient is as main density gradient material, with cotton seed
The mixing of sugar, trehalose, lactobionic acid potassium, hydroxyethyl starch one or more ingredient is as the object for preventing tissue and edema
Matter adds Magnevist Solution, can with one of HBSS, Hanks liquid or one of its improved solution for solvent,
Prepare obtained a kind of tissue and cell density gradient separations solution.
The quality of density gradient can be by evaluating its solution quality, tissue and cell separative efficiency.
The tissue and cell transplantation of the big animal such as dog or macaque or people often first need to carry out the separation of tissue and cell, close
Spending gradient centrifugation is method more common in these separation methods again, such as the separation of islet tissue.If can obtain a large amount of high-purity
The good islet cells of degree, activity will play an important role to its transplanting in vivo and raising treating diabetes effect.This patent is declared
For the exploitation of the density gradient separation liquid and applied to laying the foundation.
Summary of the invention
It is continued to develop for current big animal or tissue and the trend of cell transplantation, the purity isolated to tissue, amount
It is also higher and higher with the requirement of activity or function, it just can guarantee better transplantation effect, therefore we devise such novel group
It knits and isolates and purifies reagent with cell, the separative efficiency of tissue and cell can be improved, make up conventional separation agent point cellifugal
The deficiency of efficiency and activity aspect studies the separation and transplanting that promote state's inner cell and tissue, has and preferably apply valence
Value.It is that the clinical cytology for carrying out the product and tissue transplantation application lay the foundation that this tissue, which separates solution with cell also,.
The invention patent to be designed to provide a kind of principle simple, preparation is easy, and easy to use, tissue is separated with cell
High-efficient, the good density gradient purification reagent of activity can be made relevant using this density gradient in conjunction with density gradient centrifugation
The different tissue of density and cell are separated.
The present invention is realized using following proposal, a kind of to organize in the formula with cell purification density gradient solution
Density gradient ingredient is one of Iodixanol, Iohexol, Sodium Amidotrizoate, metrizamide, colloidal silicon, ficoll 400 or several
Kind of group is combined into, using gossypose, trehalose, lactobionic acid potassium, hydroxyethyl starch one or more combination as raising osmotic pressure and
Prevent the substance of tissue and edema, solvent composition DMEM, RPMI 1640, CMRL 1066, in M199 culture solution
A kind of one of or its improved solution.
A kind of tissue and the density gradient ingredient in the formula of cell purification density gradient solution are Iodixanol, iodine sea
One or more of alcohol, Sodium Amidotrizoate, metrizamide, colloidal silicon group is combined into, with gossypose, trehalose, lactobionic acid potassium, hydroxyl
The combination of one or more of hydroxyethyl starch is as the substance for improving osmotic pressure with preventing tissue and edema, solvent composition
For one of HBSS, Hanks liquid or one of its improved solution.
It is a kind of to organize also to contain Magnevist Solution in the formula with cell purification density gradient solution.
Each ingredient of density gradient is usually close in the formula of a kind of described tissue and cell purification density gradient solution
Spending concentration used in gradient liquid is Iodixanol 5%-70%, Iohexol 3%-75%, the Portugal Sodium Amidotrizoate 3%-75%, Jia Fan
Amine 3%-75%, colloidal silicon 3%-75%, ficoll 4003%-75%.
Raising osmotic pressure in the formula of a kind of described tissue and cell purification density gradient solution and prevent tissue
The ingredient usually concentration used in density gradient with edema is gossypose 0.5%-50%, trehalose 0.5%-
55%, lactobionic acid potassium 0.5%-35%, hydroxyethyl starch 0.1%-30%.
A kind of tissue and Magnevist Solution concentration in the formula of cell purification density gradient solution can be 0.1%-65%.
Solvent composition in the formula of a kind of described tissue and cell purification density gradient solution can for DMEM,
One of solution after one of RPMI1640, CMRL 1066, M199 culture solution or its improvement, if solvent is respectively 0.5-5
The DMEM solution of times concentration, 1640 solution of RPMI of 0.5-5 times of concentration, 1066 solution of CMRL of 0.5-5 times of concentration, 0.5-5
The M199 solution of times concentration.
Solvent composition in the formula of a kind of described tissue and cell purification density gradient solution can for HBSS,
One of solution after one of Hanks liquid or its improvement, if solvent is respectively the HBSS of 0.5-5 times of concentration, 0.5-5 times dense
The Hanks liquid of degree.
A kind of described tissue and cell purification with density gradient solution preparation method generally by density gradient ingredient,
It improves osmotic pressure and prevents the ingredient and Magnevist Solution of tissue, edema, be dissolved in corresponding solvent, filtration sterilization is
.
The present invention is by adopting the above scheme, by Iodixanol, Iohexol, Sodium Amidotrizoate, metrizamide, colloidal silicon, gather
The combination of one or more of sucrose 400 improves osmotic pressure and prevents edema ingredient gossypose, trehalose, lactobionic acid
One or more of potassium, hydroxyethyl starch combination, add Magnevist Solution, in conjunction with DMEM, RPMI 1640, CMRL1066,
One of solution, is prepared into a kind of organize and the separatory density gradient of cell after one of M199 culture solution or its improvement
Liquid.
The present invention is by adopting the above scheme, by Iodixanol, Iohexol, Sodium Amidotrizoate, metrizamide, colloidal silicon
One or more combination improves osmotic pressure and prevents edema ingredient gossypose, trehalose, lactobionic acid potassium, hydroxyethyl starch
One or more of combination, Magnevist Solution is added, in conjunction with after one of HBSS, Hanks liquid or its improvement in solution
One kind, be prepared into it is a kind of tissue and the separatory density gradient of cell.
The principle of the invention is simple, and preparation is easy, easy to use, and tissue and cell separating resulting are ideal, can reach scale
Batch production is played and promotes tissue and cell separation, and clinical and scientific research field cell transplantation is promoted to study application.
Detailed description of the invention
During attached drawing 1 is 2 higher density fluid of embodiment and the progress density gradient mixing of 3 low density liquid of embodiment, use
The 15ml centrifuge tube every 1 minute density gradient mixed liquor collected, the mixed liquor density gradient change curve measured.
Specific embodiment
Of the invention a kind of tissue and cell purification density gradient solution, by adopting the above scheme, by Iodixanol,
Iohexol, Sodium Amidotrizoate, metrizamide, colloidal silicon, ficoll 400, improve osmotic pressure and prevent edema ingredient gossypose,
Trehalose, lactobionic acid potassium, hydroxyethyl starch, add Magnevist Solution, in conjunction with DMEM, RPMI 1640, CMRL 1066,
One kind of solution, preparation method are general after HBSS, Hanks, M199 culture solution or HBSS, Hanks liquid one of which or improvement
For by density gradient ingredient, improve osmotic pressure and prevent edema ingredient and Magnevist Solution, be dissolved in corresponding solvent,
Filtration sterilization is up to a kind of tissue and the separatory density gradient of cell.
Embodiment 1
Density gradient formula of liquid used in a kind of tissue and cell purification process of the embodiment of the present invention 1 is as follows:
(1) it is formulated one: Iodixanol 248.0g, lactobionic acid potassium 15g, Magnevist Solution 5g, RPMI1640 are settled to 1L.
(2) preparation process:
1) each ingredient of RPMI 1640 (ingredient of RPMI 1640 can refer to the following table 1) is taken, is first carried out with 80% tri-distilled water
Dissolution, is first dissolved with HCl tune acid when necessary, then is carried out that pH value is adjusted to neutralize with NaOH, then be settled to 1L with tri-distilled water.
Each component list of table 1RPMI 1640
2) Iodixanol 248.0g, lactobionic acid potassium 15g, Magnevist Solution 5g are accurately weighed, until after 1L sterilizing in vial,
It is dissolved with above-mentioned 1640 solution of RPMI of 800ml, then is settled to 1L with above-mentioned 1640 solution of RPMI.When necessary with 1N HCl or
NaOH tune pH to 7.2 ± 0.2 is 1.132 ± 0.001g/ml with hand-held electronic densitometer measurement density, if it is not, can
It is a small amount of that Iodixanol or RPMI1640 progress density adjustment is added.
3) with 0.22 μm of filtering with microporous membrane after A grades of part clean area first uses 0.45 μm under B grades of backgrounds, 500mL/ bottles.
(3) it is formulated two: Iodixanol 248.0g, lactobionic acid potassium 15g, RPMI1640 are settled to 1L.
(4) preparation process: preparation process is the same as formula one.With hand-held electronic densitometer measurement density be 1.132 ±
0.001g/ml, if it is not, Iodixanol or RPMI1640 progress density adjustment can be added on a small quantity.
(5) quality evaluation of density gradient
It 1) whether is 310-360mOsm/kg with the osmotic pressure of osmotic tester measurement solution；In endotoxin measurement determines
Content of toxins whether < 0.25EU/mL.
2) bottle cap is covered tightly, and is sealed with plastic seal epiphragma；Bacteria cultivation results are sterile.
(6) method of islet tissue separation
1) the islet tissue 20ml after digestion and washing is suspended in 4 DEG C using gradient of continuous density centrifugal method
In RPMI1640 solution and it is settled to 100ml.
2) higher density fluid: using the density of above-mentioned preparation for the density gradient solution of 1.132 ± 0.001g/ml about 470ml,
With commercially available organ preservative fluid UW liquid 4 DEG C or so dilute, obtain density be 1.10 ± 0.001g/ml higher density fluid about
500ml。
3) low density liquid: using commercially available Optiprep solution about 30ml, and being diluted to density at 4 DEG C or so with UW liquid is 1.06
The low density liquid of ± 0.01g/ml, in total about 250ml.
4) higher density fluid of 120-130ml is first directed into using peristaltic pump by the cell in 2991 separator cell of COBE
It purifies in bag, opens separator cell, 2000rpm removes the bubble in cell purification bag.
5) density gradient mixer is used, above-specified high density liquid and low density liquid are respectively placed in two buckets, then is beaten
The connector among two buckets is opened, low density liquid is slowly flow in higher density fluid and mixed by connector, then passes through wriggling
Pump is directed into ultracentrifugal cell purification bag.
Etc. 6) after whole density gradients is directed into cell separation bag, then it is gradually that the liquid containing islet tissue is slow
It is directed into cell separation bag, is distributed in islet tissue from outer secretory tissue in different density gradient liquid layers, reaches separation
Purpose.
7) isolated pancreas islet is gradually collected into the different collecting pipes containing CMRL 1066.Carry out quality after purification
It examines.
(5) result of Islet purification
1) the pancreas islet rate of recovery: the people's islet tissue for 30% purity of > that collection purifying obtains, the pancreas islet rate of recovery=after purification
Pancreas islet equivalent/pancreas islet equivalent * 100% before purification；The pancreas islet rate of recovery that formula one obtains is formulated two and is free of up to 70.6%
The density gradient pancreas islet rate of recovery of Magnevist Solution is 51.4%, illustrates that the density gradient pancreas islet after Magnevist Solution is added to return
Yield obviously increases.
2) activity of pancreas islet: FD-PI dyeing is carried out using islet tissue after purification, checks pancreas islet under fluorescence microscope
Activity, fluoresced green is living cells, and sending out red fluorescence is dead cell, and the volume ratio of entire cell mass is accounted for living cells
Rate defines the activity of islet cells group, and being as a result formulated an obtained pancreas islet average activity is 88.7%, the pancreases that formula two obtains
Island average activity is 86.8%, illustrates that the islet viability that the density gradient of two formulas obtains is close.
Embodiment 2
A kind of tissue of the embodiment of the present invention 2 and highdensity density gradient formula of liquid during cell purification are as follows:
(1) be formulated: Iodixanol 204.0g, Sodium Amidotrizoate 7.36g, Magnevist Solution 10.0g, CMRL 1066 are settled to
1L。
(2) preparation process:
1) each ingredient of CMRL 1066 (ingredient of CMRL 1066 can refer to the following table 2) is taken, is first carried out with 80% tri-distilled water
Dissolution, is first dissolved with HCl tune acid when necessary, then is carried out that pH value is adjusted to neutralize with NaOH, then be settled to 1L with tri-distilled water.
Each component list of table 2CMRL 1066
2) Iodixanol 204.0g, Sodium Amidotrizoate 7.36g, Magnevist Solution 10.0g are accurately weighed, until glass after 1L sterilizing
In bottle, dissolved with above-mentioned 1066 solution of CMRL of 800ml, then be settled to 1L with above-mentioned 1066 solution of CMRL.1N is used when necessary
HCl or NaOH tune pH to 7.2 ± 0.2 is 1.10 ± 0.01g/ml with hand-held electronic densitometer measurement density, if not provided,
Iodixanol, Sodium Amidotrizoate or CMRL 1066 can then be added on a small quantity and carry out density adjustment.
3) with 0.22 μm of filtering with microporous membrane after A grades of part clean area first uses 0.45 μm under B grades of backgrounds, 500mL/ bottles.
(3) quality evaluation of density gradient
It 1) whether is 410-460mOsm/kg with the osmotic pressure of osmotic tester measurement solution；In endotoxin measurement determines
Content of toxins whether < 0.25EU/mL.
2) bottle cap is covered tightly, and is sealed with plastic seal epiphragma；Bacteria cultivation results are sterile.
Embodiment 3
A kind of tissue of the embodiment of the present invention 3 and the density gradient formula of liquid of the low-density during cell purification are as follows:
(1) be formulated: Iodixanol 10.22g, Sodium Amidotrizoate 3.59g, Magnevist Solution 15g, CMRL 1066 are settled to
250ml。
(2) preparation process:
1) Iodixanol 10.22g, vial after Sodium Amidotrizoate 3.59g, Magnevist Solution 15g to 1L sterilizing are accurately weighed
In, it is dissolved with above-mentioned 1066 solution of CMRL of 200ml, then be settled to 250ml with above-mentioned 1066 solution of CMRL.1N is used when necessary
HCl or NaOH tune pH to 7.2 ± 0.2 is 1.06 ± 0.01g/ml with hand-held electronic densitometer measurement density, if not provided,
Iodixanol, Sodium Amidotrizoate or CMRL 1066 can then be added on a small quantity and carry out density adjustment.
3) with 0.22 μm of filtering with microporous membrane after A grades of part clean area first uses 0.45 μm under B grades of backgrounds, 500mL/ bottles.
(3) quality evaluation of density gradient
It 1) whether is 450-530mOsm/kg with the osmotic pressure of osmotic tester measurement solution；In endotoxin measurement determines
Content of toxins whether < 0.25EU/mL.
2) bottle cap is covered tightly, and is sealed with plastic seal epiphragma；Bacteria cultivation results are sterile.
(4) method of islet tissue separation
1) the islet tissue 20ml after digestion and washing is suspended in 4 DEG C using gradient of continuous density centrifugal method
In RPMI1640 solution and it is settled to 100ml.
2) higher density fluid: with peristaltic pump by the density for using above-described embodiment 2 to prepare for the density of 1.10 ± 0.01g/ml
Gradient solution about 125ml is first directed into the cell purification bag in 2991 separator cell of COBE, opens separator cell,
2000rpm removes the bubble in cell purification bag.
5) density gradient mixer is used, 2 higher density fluid of above-described embodiment and 3 low density liquid of embodiment are respectively placed in
In two buckets of density gradient mixer, then the connector among two buckets is opened, low density liquid is slowly flowed by connector
It in high density liquid and mixes, then is directed into ultracentrifugal cell purification bag by peristaltic pump.
Etc. 6) after whole density gradients is directed into cell separation bag, then it is gradually that the liquid containing islet tissue is slow
It is directed into cell separation bag, is distributed in islet tissue from outer secretory tissue in different density gradient liquid layers, reaches separation
Purpose.
7) isolated pancreas islet is gradually collected into the different collecting pipes containing CMRL 1066, carries out quality after purification
It examines.
(5) result
1) 2 higher density fluid of above-described embodiment and 3 low density liquid of embodiment density gradient result: are subjected to density gradient mixing
During, with 15ml centrifuge tube every 1 minute one pipe density gradient mixed liquor of collection, its density is measured, obtained density gradient
Change curve is as shown in Figure 1, as shown in Figure 1, in higher density fluid and low density liquid mixed process, density be from high to low gradually under
Drop, the initial density of mixed liquor close to higher density fluid, terminal density close to low density liquid, be conducive in this way group be woven in it is each
Density layer finds respectively suitable density layer, and then is conducive to the separation of tissue.
2) it the islet tissue rate of recovery and activity: collects and obtains people's islet tissue of different purity, the rate of recovery of pancreas islet can
Up to 72.5%；Activity is up to 89.3%.
Claims (8)
1. a kind of tissue and cell purification density gradient solution, it is characterised in that: density gradient ingredient therein is iodine gram sand
One or more of alcohol, Iohexol, Sodium Amidotrizoate, metrizamide, colloidal silicon, ficoll 400 are formed at subassembly, with cotton seed
One or more of sugar, trehalose, lactobionic acid potassium, hydroxyethyl starch at subassembly as improve osmotic pressure and prevent tissue and
The substance of edema, one of solvent composition DMEM, RPMI 1640, CMRL 1066, M199 culture solution or its change
One of solution after；
Also contain Magnevist Solution 0.1%-65%.
2. tissue according to claim 1 and cell purification density gradient solution, it is characterised in that: wherein density gradient
The concentration that each ingredient uses is Iodixanol 5%-70%, Iohexol 3%-75%, Sodium Amidotrizoate 3%-75%, metrizamide
3%-75%, colloidal silicon 3%-75%, ficoll 4003%-75%；It wherein improves osmotic pressure and prevents tissue and edema
Ingredient concentration used in density gradient be gossypose 0.5%-50%, trehalose 0.5%-55%, lactobionic acid potassium
0.5%-35%, hydroxyethyl starch 0.1%-30%.
3. tissue according to claim 1 and cell purification density gradient solution, it is characterised in that: the solvent composition
For one of one of DMEM, RPMI 1640, CMRL 1066, M199 culture solution or its improved solution；It is described molten
Agent is respectively the DMEM solution of 0.5-5 times of concentration, the RPMI1640 solution of 0.5-5 times of concentration, the CMRL of 0.5-5 times of concentration
The M199 solution of 1066 solution or 0.5-5 times of concentration.
4. it is according to claim 1 tissue and cell purification density gradient solution, it is characterised in that: by density gradient at
Divide, improve osmotic pressure and the ingredient of tissue and edema, Magnevist Solution is prevented to be dissolved in corresponding solvent, filtration sterilization
To obtain the final product.
5. a kind of tissue and cell purification density gradient solution, it is characterised in that: density gradient ingredient therein is iodine gram sand
One or more of alcohol, Iohexol, Sodium Amidotrizoate, metrizamide, colloidal silicon are formed at subassembly, with gossypose, trehalose,
One or more of lactobionic acid potassium, hydroxyethyl starch are at subassembly as improving osmotic pressure and prevent tissue and edema
Substance, one of solvent composition HBSS, Hanks liquid or one of its improved solution；Also contain the Portugal Ga Pensuan
Amine 0.1%-65%.
6. tissue according to claim 5 and cell purification density gradient solution, it is characterised in that: wherein density gradient
The concentration that each ingredient uses is Iodixanol 5%-70%, Iohexol 3%-75%, Sodium Amidotrizoate 3%-75%, metrizamide
3%-75%, colloidal silicon 3%-75%；It wherein improves osmotic pressure and prevents tissue and the ingredient of edema in density gradient
Used in concentration be gossypose 0.5%-50%, trehalose 0.5%-55%, lactobionic acid potassium 0.5%-35%, hydroxyethyl starch
0.1%-30%.
7. tissue according to claim 5 and cell purification density gradient solution, it is characterised in that: the solvent composition
It can be one of one of HBSS, Hanks liquid or its improved solution；The solvent is the HBSS of 0.5-5 times of concentration
Or the Hanks liquid of 0.5-5 times of concentration.
8. it is according to claim 5 tissue and cell purification density gradient solution, it is characterised in that: by density gradient at
Divide, improve osmotic pressure and the ingredient of tissue and edema, Magnevist Solution is prevented to be dissolved in corresponding solvent, filtration sterilization
To obtain the final product.
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| CN109468266A (en) * | 2018-11-26 | 2019-03-15 | 温州医科大学 | Tissue, cell washing solution and preparation method after animal and the digestion of human pancreas' organ |
| CN110283782A (en) * | 2019-06-27 | 2019-09-27 | 昆明医科大学第二附属医院 | A kind of method for building up of human marrow mesenchymal stem cell strain and induction differentiation application |
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| CN103667174A (en) * | 2012-09-07 | 2014-03-26 | 北京东方华辉生物医药科技有限公司 | Composition for processing biological sample |
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