CN104922059A - Umbilical cord mesenchymal stem cell injection and preparation method and application thereof - Google Patents
Umbilical cord mesenchymal stem cell injection and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to umbilical cord mesenchymal stem cell injection and a preparation method and application thereof. The injection comprises umbilical cord mesenchymal stem cells and mixed solution; the mixed solution comprises balanced electrolyte solution, hydroxyethyl starch, adenosine disodium triphosphate-magnesium chloride, dimethyl sulfoxide and human serum albumin. The preparation method comprises the following steps: (1) fully and evenly mixing the balanced electrolyte solution, the hydroxyethyl starch and the human serum albumin, and performing filtration sterilization; (2) adding the umbilical cord mesenchymal stem cells; (3) adding the dimethyl sulfoxide; (4) performing split charging and frozen preservation to finish preparation; the invention also provides the application of the injection in the aspect of drugs for treating chronic ischemic heart diseases. The injection disclosed by the invention has a very good resuscitation effect after the frozen preservation, can be directly injected after being unfrozen and resuscitated, and has a remarkable effect on treatment on the chronic ischemic heart diseases.
Description
Technical field
The present invention relates to biomedicine field, be specifically related to a kind of umbilical cord mesenchymal stem cells injection and its preparation method and application.
Background technology
Ischemic heart desease (ischemic heart disease, IHD) is the cardiac damage caused because coronary artery circulation change causes imbalance between coronary flow and myocardial cell demand.Although in recent years, percutaneous coronary intervention (pci) (PCI), bypass operation of coronary artery (CABG) and Drug therapy achieve significant progress, but ischemic heart desease remains the worldwide No.1 cause of the death, there is the case of 20% because Coronary Artery Lesions is serious or fill the air, routine medication poor effect and cannot operative treatment, IHD sufferer is also referred to as " without right to choose (No-option) " patient.The clinical treatment mode of chronic ischemic heart disease is limited, curative effect and prognosis poor, need to find more effective novel therapeutic mode.Recently, experiment and clinical research show, stem-cell therapy IHD has a high potential, but great majority research focuses mostly in acute ischemia field, and chronic ischemic heart disease research is less.
In recent years, the progress of stem-cell research is that multiple chronic and treatment that is stubborn disease provides new strategy.In view of security consideration, current mescenchymal stem cell safety record before clinical and in clinical research is good, has had part disease such as graft versus host disease (GvHD) that mescenchymal stem cell (MSC) is introduced clinical treatment.MSC can derive from Various Tissues, and MSC conventional is at present derived from bone marrow.There are some researches show, the MSC (BM-MSCs) of derived from bone marrow may be used for the reparation of heart and injury, and the result of clinical research also demonstrates huge prospect.Multiple preclinical study shows, can improve cardiac function and reduce infarct size after BM-MSCs treatment.But also studies have found that, advanced age and disease can affect the quality and quantity of stem cell, the heart effect that the stem cell for the treatment of old people is used for the treatment of damage is poorer than the youthful stem cell for the treatment of.In addition, the risk with traumatic, virus contamination of bone marrow extraction and limited bone marrow donor limit the application of BM-MSCs.Had research to adopt other sources as fatty tissue at present, umbilical cord tissue derived mesenchymal stem cell, also achieves good effect.Umbilical cord China Tong Shi tissue has abundant MSCs, and has the features such as not damaged of drawing materials, cell yield is high, multiplication capacity is strong.There are some researches show, umbilical cord China Tong Shi glue source MSC (WJ-MSCs) has the potential being divided into various kinds of cell, comprises myocardial cell.
Current umbilical cord mesenchymal stem cells preparation is prepared through digestion mainly through neonatal umbilical cord, but large-scale demand needs more high efficiency preparation method, at present for heart class disease research with use in the public technology of umbilical cord mesenchymal stem cells have: CN 101690731 B umbilical cord Wharton jelly source mescenchymal stem cell is preparing the application in the heart failure cellular transplantation material caused because of coronary heart disease and DCM (dilated cardiomyopathy), the invention provides and fresh preparation or the mescenchymal stem cell after liquid nitrogen cryopreservation process are again cultivated after recovery, the method of carrying out infusion or injection is mixed again with sodium chloride injection etc., but namely the method declines to a great extent in 8-24 hours later cell activity, cannot ensure cytoactive for a long time, also need the mechanism carrying out treating to set up aseptic Cell Culture Center in addition and prepare personnel with the cell culture of specialty, treat each time and all want single culture, the cell quality disunity that the difference in operation of different operating personnel is brought, and the risk increasing that microbiological contamination pollutes.CN 102908364A discloses a kind of mesenchymal stem cell injection and preparation method thereof and the application in preparation treatment child dilated cardiomyopathy medicine, mesenchymal stem cell injection contains: 2 × 10
5-1 × 10
7the mescenchymal stem cell of individual/mL, volume ratio is human albumin and the compound electrolyte solution of the clinical grade DMSO of 5-8%, 1-6%.
Cell therapy approach and therapeutic effect closely related.The transplanting mode the most often adopted for stem-cell therapy at present has three kinds, is respectively in cardiac muscle, arteria coronaria and intravenous injection.Often kind of transplanting mode is all both advantageous and disadvantageous, and intramyocardial injection by cell direct injection to the cardiac muscle of damage, can improve cell implant region rate, but it can require complicated equipment and superb operation skill.Intracoronary injection wound is little, and is widely used in clinical, easy to operate, but Cellular retention rate declines to have report to think.The quality of intramyocardial injection and intracoronary injection is still disputable at present.Intravenous injection is widely adopted in the clinical research of stem-cell therapy, and simple and safe, but in cardiac disease treatment, has research to think and is mostly trapped in pulmonary after intravenous injection, so be difficult to heart of going back to the nest, can not play therapeutic effect.But studies have found that, vein transplantation cell also can improve cardiac function and reduce infarct size, and think that the TSG6 that this may secrete after lung tissue with Cellular retention is relevant.TSG6 can promote the reparation of heart and injury, but single administering mode cannot meet the complicated ischemic heart desease state of an illness, therefore in order to improve therapeutic effect further, takes new various ways administering drug combinations urgently to break through.
Summary of the invention
An object of the present invention is umbilical cord mesenchymal stem cells injection providing a kind of frozen rear resuscitation effect splendid and its preparation method and application;
Two of object of the present invention be to provide a kind of can for the preparation of umbilical cord mesenchymal stem cells injection for the treatment of chronic ischemic heart disease medicine and its preparation method and application.
First aspect, the invention provides a kind of umbilical cord mesenchymal stem cells injection, umbilical cord mesenchymal stem cells and mixed solution, described mixed solution comprises: balanced electrolyte solution, hetastarch, adenosine triphosphate disodium salt-magnesium chloride, dimethyl sulfoxide and human albumin.
Wherein, balanced electrolyte solution and human albumin maintain crystalloid osmotic pressure and colloid osmotic pressure respectively, make cell be in good osmotic pressure environment, are beneficial to growth; Dimethyl sulfoxide (DMSO) is routine clinical use cell cryopreservation protective agent; Hetastarch can make surface of cell membrane negative charge, prevents cell aggregation, also can make the depolymerization of gathering cell, thus makes cell be single released state, is conducive to improving viability rate, does not block utensil when ensureing infusion; Adenosine triphosphate-disodium magnesium chloride can provide energy directly to mescenchymal stem cell, improves the rear survival rate of recovery and cell state.The mixing of described component can improve the effect after the motility rate of freeze-stored cell and infusion, forms frozen-infusion integration solution, can after recovery of thawing direct injection and excellent, eliminate the time of wait and the operation of process.
Preferably, described balanced electrolyte solution is Bomaili A, sodium acetate ringer injection, the mixed solution of any one or at least two kinds in sodium lactate ringer's injection or inverted sugar electrolytes injection, it can be such as Bomaili A, sodium acetate ringer injection, the mixed solution of sodium lactate ringer's injection and inverted sugar electrolytes injection, sodium acetate ringer injection, the mixed solution of sodium lactate ringer's injection and inverted sugar electrolytes injection, or Bomaili A, sodium acetate ringer injection, the mixed solution of sodium lactate ringer's injection and inverted sugar electrolytes injection, balanced electrolyte solution selects according to different sufferer physical condition and to the medication taboo of contained material.Wherein, containing having sodium chloride 5.26g, gluconic acid sodium salt 5.02g, sodium acetate 3.68g in every 1000mL Bomaili A injection, potassium chloride 0.37g and magnesium chloride 0.30g; Containing sodium chloride 6.0g, sodium acetate 3.8g, potassium chloride 0.30g and calcium chloride 0.20g in every 1000mL sodium acetate ringer injection; Containing sodium lactate 3.10g, sodium chloride 6.00g, potassium chloride 0.30g and calcium chloride 0.20g in every 1000mL sodium lactate ringer's injection; Containing glucose 50g, fructose 50g, sodium chloride 1.46g, potassium chloride 1.86g, magnesium chloride 0.286g, sodium dihydrogen phosphate 0.75g and sodium lactate 2.80g in every 1000mL inverted sugar electrolytes injection.
Preferably, described hetastarch is hetastarch 130/0.4, and its chemical name is poly-(oxygen-2-ethoxy) starch 130/0.4, for clinical practice blood volume expands agent, has good cytoprotection.
Preferably, and described adenosine triphosphate disodium salt-magnesium chloride (chemical name ribosidoadenine-5 '-triguaiacyl phosphate disodium salt) be freeze dried powder, i.e. aseptic freeze-dried product, every bottle containing adenosine triphosphate disodium salt 100mg and magnesium chloride 32mg; Adenosine triphosphate disodium salt-magnesium chloride not only directly can provide energy to the cell after cryopreservation resuscitation, increases the motility rate after cell recovery, maintains the homergy ability of cell; After focus in-situ injection or venoclysis, primary power when mescenchymal stem cell is assembled at culprit lesion place can also be supported, be increased in sufferer place mescenchymal stem cell aggregate amount, accelerate the reparation speed of focus; In addition, mescenchymal stem cell utilizes paracrine action also can drive autologous stem cells reparation when sufferer place repairs, need Power supply in this process simultaneously, and adenosine triphosphate disodium salt-magnesium chloride can play and as above acts in repeatedly infusion process.Preferably, described dimethyl sulfoxide is clinical grade.
Described human albumin is the human blood albumin products of States Pharmacopoeia specifications; Preferably, the concentration of the human albumin that the present invention limits is 10-25%, can be such as 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%, be preferably 20%.
Preferably, the concentration of described umbilical cord mesenchymal stem cells is 5 × 10
5-1 × 10
6individual/mL can be such as 5 × 10
5individual/mL, 5.5 × 10
5individual/mL, 6 × 10
5individual/mL, 6.5 × 10
5individual/mL, 7 × 10
5individual/mL, 7.5 × 10
5individual/mL, 8 × 10
5individual/mL, 8.5 × 10
5individual/mL, 9 × 10
5individual/mL, 9.5 × 10
5individual/mL or 1 × 10
6individual/mL.
Preferably, described mixed solution comprises following component:
Volumn concentration is the balanced electrolyte solution of 25-70%;
Volumn concentration is the dimethyl sulfoxide of 5-20%;
Volumn concentration is the human albumin of 1-50%;
Be the hetastarch of 1-10% by the quality percent by volume of g/mL; With
By adenosine triphosphate disodium salt-magnesium chloride that the quality percent by volume of g/mL is 5-20%.
Wherein, the volume fraction of balanced electrolyte solution can be 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%; The volume fraction of dimethyl sulfoxide can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%; The volume fraction of human albumin can be 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%; The quality percent by volume of hetastarch can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%; The quality percent by volume of adenosine triphosphate disodium salt-magnesium chloride can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.
Preferably, described mixed solution comprises following component:
Volumn concentration is the balanced electrolyte solution of 30-60%;
Volumn concentration is the dimethyl sulfoxide of 10-20%;
Volumn concentration is the human albumin of 10-40%;
Be the hetastarch of 5-10% by the quality percent by volume of g/mL; With
By adenosine triphosphate disodium salt-magnesium chloride that the quality percent by volume of g/mL is 10-20%.
Preferably, described mixed solution comprises following component:
Volumn concentration is the balanced electrolyte solution of 34-50%;
Volumn concentration is the dimethyl sulfoxide of 10%;
Volumn concentration is the human albumin of 20-40%;
Be the hetastarch of 6% by the quality percent by volume of g/mL; With
By adenosine triphosphate disodium salt-magnesium chloride that the quality percent by volume of g/mL is 10%.
Preferably, described mixed solution comprises following component:
Volumn concentration is the balanced electrolyte solution of 44%;
Volumn concentration is the dimethyl sulfoxide of 10%;
Volumn concentration is the human albumin of 30%;
Be the hetastarch of 6% by the quality percent by volume of g/mL; With
By adenosine triphosphate disodium salt-magnesium chloride that the quality percent by volume of g/mL is 10%.
Preferably, the preparation method of described umbilical cord mesenchymal stem cells comprises the following steps:
(1) get the fresh umbilical cord of people, after cleaning, shear in bulk;
(2) impurity is removed, cleaning after chopping again;
(3) be immersed in digestion solution and digest, described digestion solution comprises Collagenase, hyaluronidase, neutral protease and D-Hanks liquid;
(4) filter, clean and add UMSC-CM2 culture medium after subpackage and be placed in CO
2cultivate in incubator;
(5) cell attachment carries out had digestive transfer culture cultivation 2-5 generation after merging; It can be such as 2 generations, 3 generations, 4 generations or 5 generations;
(6) washing and resuspended rear for subsequent use.
Preferably, described cleaning is for clean with D-Hanks liquid.
Each constituent content the present invention for Digestive system does not make concrete restriction, those skilled in the art can rule of thumb select, and the volume fraction of such as Collagenase can be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%; The volume fraction of hyaluronidase can be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%; The volume fraction of neutral protease can be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%; The volume fraction of D-Hanks liquid can be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.As optimal technical scheme, described digestion solution by volume mark comprises the D-Hanks liquid of the Collagenase of 5-20%, the hyaluronidase of 1-10%, the neutral protease of 1-10% and 60-90%;
More preferably, described digestion solution by volume mark comprise 10% Collagenase, the hyaluronidase of 5%, the neutral protease of 5% and 80% D-Hanks liquid.
Preferably, step (4) described cultivation, for being cultured to replaced medium after cell attachment, was carried out changing liquid every 2-4 days.
Preferably, step (5) described digestion is for carry out with serum-free trypsin.
Second aspect, the invention provides the preparation method of umbilical cord mesenchymal stem cells injection as described in relation to the first aspect, comprises the following steps:
(1) balanced electrolyte solution, hetastarch and human albumin are fully mixed, filtration sterilization;
(2) umbilical cord mesenchymal stem cells is added;
(3) dimethyl sulfoxide is added;
(4) subpackage is frozen, prepares complete.
For the addition of umbilical cord mesenchymal stem cells, the present invention does not make concrete restriction, but its final concentration will be kept to be 5 × 10
5-1 × 10
6individual/mL; Typical case but without limitation, addition conventional in the present invention is 10mL, 20mL and 50mL, and those skilled in the art can select according to practical situation.
Preferably, described filtration sterilization adopts degerming level hydrophilic filter to carry out, be preferably the degerming level hydrophilic filter of aperture 0.1-1 μm, described aperture can be such as 0.1 μm, 0.22 μm, 0.45 μm, 0.66 μm, 0.75 μm or 0.98 μm, is more preferably the degerming level hydrophilic filter in 0.22 μm, aperture.
Obtained umbilical cord mesenchymal stem cells injection can use immediately according to clinical needs, also programmed cooling can be carried out by service routine cooling instrument, be placed in liquid nitrogen storage and transport, need the 37 DEG C of water-baths putting into preheating when using to carry out recovery of thawing, direct infusion.For injection of the present invention, preferred injection is arteria coronaria-combined with intravenous input method, after specifically patient being anaesthetized, takes artery of extremity intubation procedure, conduit is delivered to left hat opening, according to 5 × 10
5the dosage slow infusion umbilical cord mesenchymal stem cells injection of individual/kg body weight.After observing one week, add twice umbilical cord mesenchymal stem cells injection (one week, interval), by vein according to 1 × 10 respectively by vein
6/ kg body weight dose infusion umbilical cord mesenchymal stem cells injection.
The third aspect, the invention provides the application of umbilical cord mesenchymal stem cells injection in the medicine of preparation treatment chronic ischemic heart disease as described in relation to the first aspect.
Compared with prior art, the present invention at least has following beneficial effect:
The invention provides and a kind ofly prepare umbilical cord mesenchymal stem cells injection for the treatment of chronic ischemic heart disease medicine and its preparation method and application.The present invention takes full advantage of umbilical cord mesenchymal stem cells without immunologic rejection, without oncogenicity and the advantage fast that increases, avoid ethics and legal issue; By taking rapid digestion cultural method, prepare umbilical cord mesenchymal stem cells in a large number rapidly, and by adopt frozen-inject integrated solution and the formulated injection of umbilical cord mesenchymal stem cells, composition is not containing animal serum, and the rear Cell viability of recovery meets treatment requirement;
While guarantee cell viability, medical safety and effectiveness, injection of the present invention also facilitates clinical direct input to use, liquid nitrogen cryopreservation can be utilized, and through thawing recovery after direct injection, solve the problem that inconvenience was implemented, transported and stored to current mescenchymal stem cell in chronic ischemic heart disease treatment, medical personnel's operation can be convenient to simultaneously, eliminate the waiting time of sufferer;
Take the mode of arteria coronaria-combined with intravenous infusion, avoid that arteria coronaria single infused cells remaining time is short, the single infusion of vein pulmonary and liver accumulation and effectively cannot reach the problem at sufferer place;
Chronic ischemic heart disease symptom and prognosis is improved by the self duplication of mescenchymal stem cell, tissue repair and paracrine action, especially a kind of novel Therapeutic Method is brought for part conventional medicine and the invalid patient of operation, reduce the mortality rate of this type of disease.
Accompanying drawing explanation
Fig. 1 is umbilical cord mesenchymal stem cells aspect graph prepared by embodiment 1;
Fig. 2 is the flow cytomery result figure of umbilical cord mesenchymal stem cells prepared by embodiment 1;
Fig. 3 is the Differentiation Induction in vitro result figure of umbilical cord mesenchymal stem cells prepared by embodiment 1;
Fig. 4 is the flow cytomery result figure of umbilical cord mesenchymal stem cells after 24 months cryopreservation resuscitations prepared by embodiment 2;
Fig. 5 is the Differentiation Induction in vitro result figure of umbilical cord mesenchymal stem cells after 24 months cryopreservation resuscitations prepared by embodiment 2;
Fig. 6 is the restitution experimental result of umbilical cord mesenchymal stem cells to chronic ischemic heart disease animal model---SDS difference results figure;
Fig. 7 is the restitution experimental result of umbilical cord mesenchymal stem cells to chronic ischemic heart disease animal model---myocardial infarction area and fibrosis measurement result figure;
Fig. 8 is for umbilical cord mesenchymal stem cells is to the restitution experimental result of chronic ischemic heart disease animal model---red fluorescence signature.
Detailed description of the invention
Technical scheme of the present invention is further illustrated below by detailed description of the invention.Those skilled in the art should understand, described embodiment is only help to understand the present invention, should not be considered as concrete restriction of the present invention.
The preparation of embodiment 1 umbilical cord mesenchymal stem cells
The present embodiment adopts following steps to carry out the preparation of umbilical cord mesenchymal stem cells:
(1) the fresh umbilical cord that the parent time of leaving of getting cesarean intercepting is no more than 2 hours, and check whether puerpera carries infectious disease pathogen (hepatitis B surface antigen, c-hepatitis antibody, syphilis antibody and aids antibody are negative), placed by fresh umbilical cord in the special preservation bottle containing D-Hanks balance liquid, low temperature ice box transports;
(2) Biohazard Safety Equipment ultra-vioket radiation 30min, shaking bath 37 DEG C preheating in advance, is placed on umbilical cord sample bottle in Biohazard Safety Equipment and operates.Taking out sample with long mosquito forceps is placed in 150mm sterile glass ware, washes away umbilical cord surface blood with D-Hanks balance liquid.
(3) umbilical cord tissue after cleaning being carried out mechanical shearing is about 2-5cm segment, and the umbilical artery in careful removal tissue and vein, peel off umbilical cord adventitia.Umbilical cord Wharton jelly is sheared and becomes about 2-3mm
3fritter, put into 50mL centrifuge tube, use D-Hanks buffer solution centrifugal, remove measurement volumes after supernatant, to calculate the total amount of required digestive enzyme.
(4) Fresh is containing the premix digestion solution of 10% Collagenase (USA Sigma), 5% hyaluronidase (USASigma), 5% neutral protease (USA Sigma) and 80%D-Hanks liquid.Umbilical cord tissue after centrifugal is transferred in the aseptic wide mouthed bottle of 100mL, add equal-volume enzymic digestion liquid, be put in 37 DEG C of water-baths, middling speed concussion digestion 2h, be filled in new 50mL centrifuge tube with 70 μm of screen clothes by the supernatant of collection afterwards, washing is centrifugation cell also.
(5) appropriate seed umbilical cord mesenchymal stem cells is transferred in culture dish or culture bottle, adds appropriate UMSC-CM2 culture medium, the adherent situation of observation of cell, after put into the CO of 37 DEG C
2cultivate in incubator, after merging state, use serum-free trypsinization until Growth of Cells, centrifugal, go down to posterity in subpackage to multiple culture bottle.
(6) go down to posterity 2-5 for rear observation of cell state, use PBS buffer solution after trypsinization, carry out qualification for subsequent use.
2-5 prepared by embodiment 1 is identified for umbilical cord mesenchymal stem cells, comprising:
(1) Microscopic observation can be attached at frosting (as shown in Figure 1) under Standard culture conditions: wherein, and A is visible cellular morphology clearly after cell separation inoculated and cultured 2d; B is the cellular morphology figure after cell separation inoculated and cultured 5d, can find out that now cell has formed more typical umbilical cord mesenchyma sample, and have obvious adherent feature.
(2) express CD105, CD73, CD90 and HLA-ABC (expression is higher than 90%), do not express CD45, CD34 and HLA-DR (expression is lower than 1%) (as shown in Figure 2); Concrete flow cytomery result is as shown in table 1:
Table 1 flow cytomery result
| Traget antibody | Positive rate (%) |
| CD34 | 0 |
| CD44 | 99.48 |
| CD45 | 0 |
| CD73 | 99.72 |
| CD90 | 99.58 |
| CD105 | 94.43 |
| HLA-DR | 0.02 |
| HLA-ABC | 96.27 |
(3) in vitro can through being induced to differentiate into chondroblast, adipose cell and osteoblast, all can at Microscopic observation to positive (as shown in Figure 3) through alkaline phosphatase staining, Alizarin red staining and oil red " 0 " dyeing: can find out that umbilical cord mesenchymal stem cells prepared by the present invention can break up to osteoblast, chondrocyte and lipoblast through suitable condition, prove that umbilical cord mesenchymal stem cells prepared by the present invention has the Multidirectional Differentiation ability of stem cell.
The standard that above qualification result is formulated with reference to " international cell therapy association (ISCT) ", provable prepared cell possesses the characteristic of mescenchymal stem cell.
The preparation of embodiment 2 umbilical cord mesenchymal stem cells injection
The present embodiment adopts following steps to carry out the preparation of umbilical cord mesenchymal stem cells injection:
(1) 50mL mixed solution is prepared, wherein, the percent by volume of balanced electrolyte solution is 44%, the quality percent by volume of hetastarch 130/0.4 is 6%, and the quality percent by volume of adenosine triphosphate disodium salt-magnesium chloride freeze dried powder is the percent by volume of the human albumin of 10% and 20% is 30%;
(2) mixed solution is fully mixed, uses the degerming level hydrophilic frit in 0.22 μm, aperture, then the umbilical cord mesenchymal stem cells of preparation is slowly added degerming after mixed solution in, concentration controls 5 × 10
5-1 × 10
6individual/mL;
(3) add clinical grade dimethyl sulfoxide, make its final volume mark be 10%;
(4) the umbilical cord mesenchymal stem cells injection of preparation is sub-packed in
in cell cryopreservation bag, prepare complete.
Kept sample by the injection be divided in frozen bag and carry out a batch detection, testing requirement endotoxin detects (gel method), antibacterial culturing, Viral diagnosis, detection of mycoplasma, hepatitis B surface antigen, c-hepatitis antibody, syphilis antibody and aids antibody and is feminine gender.
The preparation of embodiment 3 umbilical cord mesenchymal stem cells injection
The present embodiment adopts following steps to carry out the preparation of umbilical cord mesenchymal stem cells injection:
(1) 50mL mixed solution is prepared, wherein, the percent by volume of balanced electrolyte solution is 25%, the quality percent by volume of hetastarch 130/0.4 is 5%, and the quality percent by volume of adenosine triphosphate disodium salt-magnesium chloride freeze dried powder is the percent by volume of the human albumin of 5% and 20% is 50%;
(2) mixed solution is fully mixed, uses the degerming level hydrophilic frit in 0.22 μm, aperture, then the umbilical cord mesenchymal stem cells of preparation is slowly added degerming after mixed solution in, concentration controls 5 × 10
5-1 × 10
6individual/mL;
(3) add clinical grade dimethyl sulfoxide, make its final volume mark be 20%;
(4) the umbilical cord mesenchymal stem cells injection of preparation is sub-packed in
in cell cryopreservation bag, prepare complete.
Kept sample by the injection be divided in frozen bag and carry out a batch detection, testing requirement endotoxin detects (gel method), antibacterial culturing, Viral diagnosis, detection of mycoplasma, hepatitis B surface antigen, c-hepatitis antibody, syphilis antibody and aids antibody and is feminine gender.
The preparation of embodiment 4 umbilical cord mesenchymal stem cells injection
The present embodiment adopts following steps to carry out the preparation of umbilical cord mesenchymal stem cells injection:
(1) 50mL mixed solution is prepared, wherein, the percent by volume of balanced electrolyte solution is 70%, the quality percent by volume of hetastarch 130/0.4 is 10%, and the quality percent by volume of adenosine triphosphate disodium salt-magnesium chloride freeze dried powder is the percent by volume of the human albumin of 20% and 20% is 1%;
(2) mixed solution is fully mixed, uses the degerming level hydrophilic frit in 0.22 μm, aperture, then the umbilical cord mesenchymal stem cells of preparation is slowly added degerming after mixed solution in, concentration controls 5 × 10
5-1 × 10
6individual/mL;
(3) add clinical grade dimethyl sulfoxide, make its final volume mark be 5%;
(4) the umbilical cord mesenchymal stem cells injection of preparation is sub-packed in
in cell cryopreservation bag, prepare complete.
Kept sample by the injection be divided in frozen bag and carry out a batch detection, testing requirement endotoxin detects (gel method), antibacterial culturing, Viral diagnosis, detection of mycoplasma, hepatitis B surface antigen, c-hepatitis antibody, syphilis antibody and aids antibody and is feminine gender.
The preparation of embodiment 5 umbilical cord mesenchymal stem cells injection
The present embodiment adopts following steps to carry out the preparation of umbilical cord mesenchymal stem cells injection:
(1) 50mL mixed solution is prepared, wherein, the percent by volume of balanced electrolyte solution is 47%, the quality percent by volume of hetastarch 130/0.4 is 1%, and the quality percent by volume of adenosine triphosphate disodium salt-magnesium chloride freeze dried powder is the percent by volume of the human albumin of 12.5% and 20% is 25.5%;
(2) mixed solution is fully mixed, uses the degerming level hydrophilic frit in 0.22 μm, aperture, then the umbilical cord mesenchymal stem cells of preparation is slowly added degerming after mixed solution in, concentration controls 5 × 10
5-1 × 10
6individual/mL;
(3) add clinical grade dimethyl sulfoxide, make its final volume mark be 12.5%;
(4) the umbilical cord mesenchymal stem cells injection of preparation is sub-packed in
in cell cryopreservation bag, prepare complete.
Kept sample by the injection be divided in frozen bag and carry out a batch detection, testing requirement endotoxin detects (gel method), antibacterial culturing, Viral diagnosis, detection of mycoplasma, hepatitis B surface antigen, c-hepatitis antibody, syphilis antibody and aids antibody and is feminine gender.
The preparation of embodiment 6 umbilical cord mesenchymal stem cells injection
The present embodiment adopts following steps to carry out the preparation of umbilical cord mesenchymal stem cells injection:
(1) 50mL mixed solution is prepared, wherein, the percent by volume of balanced electrolyte solution is 30%, the quality percent by volume of hetastarch 130/0.4 is 8%, and the quality percent by volume of adenosine triphosphate disodium salt-magnesium chloride freeze dried powder is the percent by volume of the human albumin of 10% and 20% is 25%;
(2) mixed solution is fully mixed, uses the degerming level hydrophilic frit in 0.22 μm, aperture, then the umbilical cord mesenchymal stem cells of preparation is slowly added degerming after mixed solution in, concentration controls 5 × 10
5-1 × 10
6individual/mL;
(3) add clinical grade dimethyl sulfoxide, make its final volume mark be 15%;
(4) the umbilical cord mesenchymal stem cells injection of preparation is sub-packed in
in cell cryopreservation bag, prepare complete.
Kept sample by the injection be divided in frozen bag and carry out a batch detection, testing requirement endotoxin detects (gel method), antibacterial culturing, Viral diagnosis, detection of mycoplasma, hepatitis B surface antigen, c-hepatitis antibody, syphilis antibody and aids antibody and is feminine gender.
Comparative example 1
Adopt the mesenchymal stem cell injection that CN 102908364 A provides.
Comparative example 2
Adopt the mesenchymal stem cell cryopreserving liquid that CN 102792947 A provides.
Comparative example 3
Adopt the mesenchymal stem cell injection prepared of conventional method: DMSO10%, hyclone FBS10% and UMSC-CM2 culture medium 80% and concentration are 5 × 10
5-1 × 10
6the umbilical cord mesenchymal stem cells of individual/mL.
Comparative example 4
Identical with embodiment 2, just do not add dimethyl sulfoxide.
Comparative example 5
Identical with embodiment 2, just do not add adenosine triphosphate disodium salt-magnesium chloride.
Function Identification:
The umbilical cord mesenchymal stem cells injection of embodiment 2 is carried out freezing through Slow-rate freezing instrument, after put into liquid nitrogen and preserve; Mescenchymal stem cell prepared by the comparative example 1-5 simultaneously getting equivalent carries out programmed cooling and to put into liquid nitrogen frozen.
The cell cryopreservation bag of 6 kinds of frozen formula more than taking out from liquid nitrogen, get three bags for often kind, carefully put into the water-bath being preheated to 37 DEG C, the corner of clamping frozen bag with tweezers slightly adjusts angle, frozen bag is heated evenly, recovered in 1-2 minute, the cell suspension got after appropriate recovery carries out the dyeing of Placenta Hominis orchid, measure the Cell viability (i.e. living cells percentage) of frozen 6 months, 12 months, 18 months and 24 months respectively, result is as described in Table 2.
Table 2 umbilical cord mesenchymal stem cells injection compares (n=3) with the frozen rear Cell viability of conventional method
As seen from Table 2, the mescenchymal stem cell of umbilical cord mesenchymal stem cells injection of the present invention after preserving recovery for a long time still keeps higher vigor, and the general cell therapy motility rate reaching national requirements requires (>=85%), after 24 months preserve, about 5% is declined when motility rate was compared with 6 months, but still keep the high motility rate of more than 90%, ensure therapeutic effect, and provide the long-term possibility of preserving and transporting.Cell viability after cryopreservation resuscitation is compared with conventional DMSO-hyclone cryopreserving liquid, and effect is more excellent, and not containing animal sources, composition is clear controlled, illustrates that it can replace traditional cells frozen storing liquid mode, and can at the rear direct infusion of recovery of thawing.The Cell viability of embodiment 2 apparently higher than comparative example 1 and comparative example 3-5, illustrate formula of the present invention can significantly improve cell cryopreservation after resuscitation effect, promote Cell viability.
Choose the preservation frozen injection of 24 months, serum-free mescenchymal stem cell culture medium is used to cultivate to the mescenchymal stem cell after recovery, observation of cell form, and carry out flow cytometry, to one-tenth fat, become cartilage and Osteoblast Differentiation, verify that its stem capacity changes, result as shown in Figure 4 and Figure 5.As can be seen from Figure 4, preserve the sample of 24 months through the result of flow cytometry and the mescenchymal stem cell phenotype of fresh preparation consistent (Fig. 2), namely CD105, CD73, CD90 and HLA-ABC expression is higher than 90%, the expression of CD45, CD34 and HLA-DR, lower than 1%, confirms long-term preservation mescenchymal stem cell Surface Phenotype and dryness without impact.Concrete outcome is as shown in table 3:
Table 3 flow cytomery result
| Traget antibody | Positive rate (%) | Traget antibody | Positive rate (%) |
| CD34 | 0.16 | CD90 | 99.86 |
| CD44 | 99.14 | CD105 | 98.93 |
| CD45 | 0.48 | HLA-DR | 0.67 |
| CD73 | 97.08 | HLA-ABC | 96.71 |
As can be seen from Figure 5, the injection of frozen 24 months carries out recovery of thawing, cultivate according to general cultural method after washing, then in vitro can through being induced to differentiate into chondroblast, adipose cell and osteoblast, through alkaline phosphatase staining, the dyeing of Alizarin red staining, oil red " 0 " and A Li Xinlan all can at Microscopic observation to positive, and the mescenchymal stem cell proving in injection remains unchanged the ability of maintenance Multidirectional Differentiation.
Umbilical cord mesenchymal stem cells is tested the restitution of chronic ischemic heart disease animal model:
Select female Ba-Ma mini pig, carry out induced anesthesia by intramuscular injection 25mg/kg ketalar and 1mg/kg.After tracheal intubation, isoflurane inhalation anesthesia maintains, and endotracheal tube is connected to respirator and controls ventilation.Breast is opened in left side, cuts off pericardium, exposes LCX.The Ameroid ring being 2.5mm by a diameter inserts the root of LCX, sews up pericardium, successively closes breast after aerofluxus.Chronic ischemic heart disease animal model is formed after 4 weeks.Duration of test, cardiac function measures assessment primarily of following methods: echocardiography cardiac function, left ventricular end diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV), and calculate Left Ventricular Ejection Fraction EF (%)=(EDV-ESV)/EDV × 100%.
Obtain two-dimensional ultrasonic image at left room long axis view mitral level, therefrom measure LVED (Left Ventricular End Systolic Dimension) and diastole internal diameter and infarcted region chamber wall thickness and heart rate HR; Coronary angiography evaluation collateral circulation; SPECT evaluates Myocardial Perfusion, property tranquillization SPECT after intravenous injection 555MBq 99mTc-MIBI, the measurements of the chest, waist and hips of being carried out image by successive ignition are rebuild, finally be divided into by image 20 sections to carry out semi-quantitative analysis, calculate tranquillization PTS (SRS) and perfusion overall score (SDS).After test, animal tissue is carried out paraffin or the embedding of OCT glue, HE dyeing, horse pine trichroism, vWF SABC, TUNEL detects, antibody-nuclear staining measuring and calculating infarct size, angiogenesis number, apoptosis number and cell field planting and differentiation; By section Microscopic observation, with IPP6.0 computed in software collagen volume fraction measuring and calculating collagen content; Cytokine-expressing is detected by round pcr.
Insert Ameroid ring after 4 weeks, anaesthetized by pig, and to right common femoral artery intubate, under X-ray, the Medtronic conduit of 6F is delivered to left hat opening, treatment group is slow infusion 30 × 10 in 5min
6individual WJ-MSCs, the isopyknic normal saline of matched group infusion.
At the 5th and the 6th week, add twice cell respectively by vein, injection group was by auricular vein infusion 30 × 10
6individual cell, matched group inputs isopyknic normal saline, and infusion velocity is 2-3mL/min.
Experimental result represents, after treating latter 28 days, matched group heart rate obviously raises, and treatment group infarcted region systole chamber wall thickness obviously increases, and end-systolic volume and end-diastolic dimension have no obvious change, and prompting cell therapy can improve remodeling ventricle.Ultrasound detection penetrates blood contrast, and matched group is than front have dropped 5.2 ± 2.5%, and treatment group increases 5.2 ± 1.7% comparatively before.
Infarcted region systolic wall thickening rate (WThF) is zero difference between two groups before the treatment, and after treatment, WJ-MSC group obviously raises, and this illustrates that WJ-MSC treatment improves infarcted region Local systolic.Collateral circulation before adopting coronary angiography evaluation treatment and after treatment, after WJ-MSC treatment, Rentrop mark significantly raises, and rises to 2.6 ± 0.2, p<0.01 from 1.3 ± 0.2; Illustrate that WJ-MSC treatment promotes the generation of collateral circulation.SDS can be used for the change of reflecting myocardium blood perfusion, and after 4 weeks, the SDS for the treatment of group is significantly higher than matched group (Fig. 6), and prompting cell therapy can improve Myocardial Perfusion.
Myocardial infarction area and fibrosis measurement result are as shown in Figure 7.(A) be TTC dyeing, white portion represents infarcted region, and redness is normal district; (B) for treatment group infarct size accounts for the percentage ratio reduction more remarkable in matched group of left room area, the infarct size (8.4% ± 1.9%) for the treatment of group significantly will be less than matched group (14.5% ± 2.0%, p<0.05).(C) treatment group fibrosis region is shown few compared with matched group, (D) for carry out sxemiquantitative statistic analysis result to fibrosis region, can find out that treatment group and matched group exist remarkable significant difference, treatment group collagen volume fraction comparatively matched group obviously reduces, the collagen volume fraction for the treatment of group is 23.1% ± 2.0%, and matched group is 44.6% ± 3.1%, the two has remarkable significant difference, and this illustrates that utilizing injection of the present invention to carry out cell therapy can reduce infarct size and myocardial fibrosis.
Before cell therapy, with CM-DiI, red fluorescence labelling (as shown in Figure 8 A) is carried out to cell, afterwards by field planting and the differentiation of Immunofluorescence test cell.After 4 weeks, the cell of red fluorescence labelling can be seen in the marginal zone for the treatment of group heart, and have sub-fraction to endothelial cell differentiation field planting can possess differentiation due (as shown in Figure 8 B) in wound area after proving mescenchymal stem cell treatment.
Clinical practice model case:
Male patient, 76 years old, main cause " repeatedly chest pain 2 years " was admitted to hospital, and outer court is diagnosed as coronary heart disease, involves three-image difference, on inspection the omnidistance diffuse lesion of LAD, the heaviest narrow place 90%; The omnidistance diffuse lesion of LCX, there is the collatoral vessel far-end blood supply of propping up from blunt edge at the heaviest narrow place 99%; RCX opening to stage casing diffuse lesion, the heaviest narrow place 90%.Be not suitable for carrying out PCI or CABG treatment through assessment, invalid by guide maximum tolerated dose Drug therapy.Before taking umbilical cord mesenchymal stem cells to treat, LVEF index is 44%, 6 minutes walking distances are 82m, and angina pectoris attacks frequency is 6-8 times/day, take arteria coronaria infusion XX cell concentration, venoclysis is taked after one week, venoclysis again every other week, treat latter 1 month, LVEF index is increased to 46%, 6min walking distance is increased to 122m, treats and is increased to 191m in latter 6 months; Treat latter one month angina pectoris attacks frequency and be down to 3-4 times/day, after 6 months, be down to 1 time/(3-4) sky.
As can be seen from the above results, easy and simple to handle when injection provided by the invention uses, without the need to special operational place, can traditional cells frozen storing liquid be replaced, can at the rear direct infusion of recovery of thawing; Take the mode of arteria coronaria-combined with intravenous administration, be applied to chronic ischemic heart disease patient, by the proliferation and differentiation effect of mescenchymal stem cell self, and the secretory volume such as anti-inflammatory factors and angiogenesis factor of perienchyma that paracrine effect drives increases, to improve life in patients, reduce mortality rate.
Applicant states, the present invention illustrates process of the present invention by above-described embodiment, but the present invention is not limited to above-mentioned processing step, does not namely mean that the present invention must rely on above-mentioned processing step and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of auxiliary element, the concrete way choice etc. of raw material selected by the present invention, all drops within protection scope of the present invention and open scope.
Claims (9)
1. a umbilical cord mesenchymal stem cells injection, comprise umbilical cord mesenchymal stem cells and mixed solution, it is characterized in that, described mixed solution comprises: balanced electrolyte solution, hetastarch, adenosine triphosphate disodium salt-magnesium chloride, dimethyl sulfoxide and human albumin.
2. injection according to claim 1, is characterized in that, described balanced electrolyte solution is the mixed solution of any one or at least two kinds in Bomaili A, sodium acetate ringer injection, sodium lactate ringer's injection or inverted sugar electrolytes injection;
Preferably, described hetastarch is hetastarch 130/0.4;
Preferably, described adenosine triphosphate disodium salt-magnesium chloride is freeze dried powder;
Preferably, described dimethyl sulfoxide is clinical grade;
Preferably, the concentration of described human albumin is 10-25%, is preferably 20%;
Preferably, the concentration of described umbilical cord mesenchymal stem cells is 5 × 10
5-1 × 10
6individual/mL;
Preferably, described mixed solution comprises following component:
Volumn concentration is the balanced electrolyte solution of 25-70%;
Volumn concentration is the dimethyl sulfoxide of 5-20%;
Volumn concentration is the human albumin of 1-50%;
Be the hetastarch of 1-10% by the quality percent by volume of g/mL; With
By adenosine triphosphate disodium salt-magnesium chloride that the quality percent by volume of g/mL is 5-20%.
3. injection according to claim 1 and 2, is characterized in that, described mixed solution comprises following component:
Volumn concentration is the balanced electrolyte solution of 30-60%;
Volumn concentration is the dimethyl sulfoxide of 10-20%;
Volumn concentration is the human albumin of 10-40%;
Be the hetastarch of 5-10% by the quality percent by volume of g/mL; With
By adenosine triphosphate disodium salt-magnesium chloride that the quality percent by volume of g/mL is 10-20%.
Preferably, described mixed solution comprises following component:
Volumn concentration is the balanced electrolyte solution of 34-50%;
Volumn concentration is the dimethyl sulfoxide of 10%;
Volumn concentration is the human albumin of 20-40%;
Be the hetastarch of 6% by the quality percent by volume of g/mL; With
By adenosine triphosphate disodium salt-magnesium chloride that the quality percent by volume of g/mL is 10%.
4. according to the injection one of claim 1-3 Suo Shu, it is characterized in that, described mixed solution comprises following component:
Volumn concentration is the balanced electrolyte solution of 44%;
Volumn concentration is the dimethyl sulfoxide of 10%;
Volumn concentration is the human albumin of 30%;
Be the hetastarch of 6% by the quality percent by volume of g/mL; With
By adenosine triphosphate disodium salt-magnesium chloride that the quality percent by volume of g/mL is 10%.
5. according to the injection one of claim 1-4 Suo Shu, it is characterized in that, the preparation method of described umbilical cord mesenchymal stem cells comprises the following steps:
(1) in bulk is sheared by after fresh for people umbilical cord cleaning;
(2) impurity is removed, cleaning after chopping again;
(3) be immersed in digestion solution and digest, described digestion solution comprises Collagenase, hyaluronidase, neutral protease and D-Hanks liquid;
(4) filter, clean and add UMSC-CM2 culture medium after subpackage and be placed in CO
2cultivate in incubator;
(5) cell attachment carries out had digestive transfer culture cultivation 2-5 generation after merging;
(6) washing and resuspended rear for subsequent use.
6. injection according to claim 5, is characterized in that, described cleaning is for clean with D-Hanks liquid;
Preferably, described digestion solution by volume mark comprise the D-Hanks liquid of the Collagenase of 5-20%, the hyaluronidase of 1-10%, the neutral protease of 1-10% and 60-90%;
Preferably, described digestion solution by volume mark comprise 10% Collagenase, the hyaluronidase of 5%, the neutral protease of 5% and 80% D-Hanks liquid;
Preferably, step (4) described cultivation, for being cultured to replaced medium after cell attachment, was carried out changing liquid every 2-4 days;
Preferably, step (5) described digestion is for carry out with serum-free trypsin.
7., according to the preparation method of the umbilical cord mesenchymal stem cells injection one of claim 1-4 Suo Shu, comprise the following steps:
(1) balanced electrolyte solution, hetastarch and human albumin are fully mixed, filtration sterilization;
(2) umbilical cord mesenchymal stem cells is added;
(3) dimethyl sulfoxide is added;
(4) subpackage is frozen, prepares complete.
8. method according to claim 7, is characterized in that, described filtration sterilization adopts degerming level hydrophilic filter to carry out, and is preferably the degerming level hydrophilic filter of aperture 0.1-1 μm, is more preferably the degerming level hydrophilic filter in 0.22 μm, aperture.
9. according to the application of the umbilical cord mesenchymal stem cells injection one of claim 1-4 Suo Shu in the medicine of preparation treatment chronic ischemic heart disease.
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