CN104873542A - Umbilical cord mesenchymal stem cell injection as well as preparation method and application thereof - Google Patents
Umbilical cord mesenchymal stem cell injection as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides umbilical cord mesenchymal stem cell injection as well as a preparation method and application thereof. The umbilical cord mesenchymal stem cell injection comprises mesenchymal stem cells and a frozen stock solution, wherein the frozen stock solution comprises the following components: a balanced electrolyte solution with the volume percentage of 25-70 percent, hydroxyethyl starch 130/0.4 with the mass/volume percentage (g/ml) of 1-10 percent, triphosadenine disodium magnesium chloride with the volume percentage of 5-20 percent, clinical-grade DMSO with the volume percentage of 5-20% and 20% human albumin with the volume percentage of 1-50 percent. The prepared umbilical cord mesenchymal stem cell injection is free of animal serum, safe, high in cell viability after cryopreservation resuscitation, can be used for treating inflammatory bowel disease, has a good effect and has no toxic or side effects.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of umbilical cord mesenchymal stem cells injection and its preparation method and application.
Background technology
Inflammatory bowel (Inflammatory Bowel Disease, IBD) be one group of idiopathic, chronic, inflammatory bowel disease state, the irreversible damage of intestinal 26S Proteasome Structure and Function can be caused, comprise two kinds of principal disease types: Crohn disease (Crohn's Disease, and ulcerative colitis (Ulcerative Colitis, UC) CD).Crohn disease pathogenesis is complicated, still unclear at present.The intestinal stenosis that the Clinical symptoms of Crohn disease comprises diarrhoea, stomachache, stenosis of bowel cause and intestinal obstruction, abscess are formed, fistulization, can with enteritis performances such as joint, eyes, skin, mouth and livers.Ulcerative colitis is a kind of agnogenic nonspecific inflammation, and pathological changes is positioned at colorectal mucosa and tela submucosa, and distribute in seriality diffusivity, majority involves rectum and sigmoid colon.The clinical manifestation of ulcerative colitis mainly comprises mucus bloody purulent stool, stomachache and diarrhoea etc., and intestinal shows outward and can relate to each system, and the most common organ involved is skin, oral cavity, eye, joint and liver and gall.Course of disease protracted course of disease, reaches even decades more than ten years, also has the probability that canceration occurs.Along with China's process of industrialization is accelerated, the sickness rate of IBD raises year by year.This disease mostly is between twenty and fifty morbidity, and complication is many and serious, has a strong impact on life and work capacity, and still lacks clinically and effectively effect a radical cure method, has caused the great attention of research worker in world wide.Current clinical treatment is mainly conceived to control Active inflammation and immunity moderation disorder, conventional medicine has salicylic acid, steroid, immunosuppressant and biological preparation etc., and the biotherapy of application as Anti-TNF Antibody McAb etc. further improves Therapeutic Method.But apply the therapeutic scheme that these improve, still have many patients without response or As time goes on decline to the reaction for the treatment of, the maintainable remission scheme of ulcerative colitis the best also only can make relapse rate reduce about 50%.Therefore, how find further the effective therapeutic scheme that makes new advances reducing patients with inflammatory bowel disease disability rate, improve patients ' life quality and then have important practical significance and larger medical demand.
Stem cell is the cell that a class has self renewal and Multidirectional Differentiation ability, adopts the various seriousness of stem-cell therapy and stubborn disease to become the focus of research both at home and abroad.Mescenchymal stem cell (mesenchymalstem cells, MSCs), except having general stem cell feature, also has reduced immunogenicity and immunoregulation effect.Therefore, this kind of cell has very wide prospect in clinical practice.Umbilical cord mesenchymal stem cells (Umbilical cord mesenchymal stem cells, UC-MSCs) compared with other MSCs originated, have that source is sufficient, noinvasive, strong and its acquisition of multiplication capacity becomes most potential treatment source of human stem cell without advantages such as ethical issuess.UC-MSCs treats the clinical practice of the various diseases such as GVHD and anti-hypomnesis in recent years, also shows that UC-MSCs has good safety and effectiveness.
The shortcoming of current technology is: indefinite due to the current pathogenesis of inflammatory bowel, there is no clear and definite specific medicament, and the course of disease is longer, very easily recurs, the treatment means object taked at present is generally control symptom, as promoted the healing of intestines pathological changes, alleviating symptoms such as suffering from diarrhoea, have blood in stool.Pass through diet control, hormone therapy, Salicylates and willow nitrogen sulphur arsenic pyridine treatment can play the effect of certain symptom management, but simultaneously the toxic and side effects brought of medicine is also very serious, life-time service easily produces medicine response reduction or invalid, concerning patient itself, also greatly reduce quality of life; Operative treatment can play certain therapeutic effect, but higher to patient's property selected, and with certain operation risk, postoperative sequela, as severe haemorrhage, intestinal obstruction, bowel perforation, fistulization, ulceration canceration etc., risk increases further.In recent years research shows, the basic occurrence cause of inflammatory bowel is the disorder of vivo immuning system, cause the generation of chronic inflammatory disease, umbilical cord mesenchymal stem cells is on the basis not having ethics morals problem, and draw materials extensively convenient, competence for added value is strong, cell state is stablized, and possess stronger immunoregulation capability, can the immunologic derangement state existed in patient body be regulated, stop inflammatory factor and immunocyte for the destruction of self intestinal tissue; By self field planting of umbilical cord mesenchymal stem cells, differentiation and paracrine action, participate in directly or drive body to repair intestinal tract, promote the recovery of pathological changes intestinal and ulcer, and the eye involved, skin, liver etc. are organized also have good repair; For the toxic and side effects that conventional treatment drug brings, as allergy, aplastic anemia, hemolytic anemia, thrombocytopenia, granulocytopenia etc., umbilical cord mesenchymal stem cells can rely on the factors such as GM-CSF, SDF-1, SCF of its secretion to urge hemopoietic function, alleviates above symptom.
Summary of the invention
For the deficiencies in the prior art, the object of the present invention is to provide a kind of umbilical cord mesenchymal stem cells injection.
For reaching this goal of the invention, the present invention by the following technical solutions:
On the one hand, the invention provides a kind of umbilical cord mesenchymal stem cells injection, described infusion pump is containing mescenchymal stem cell and cryopreserving liquid, and wherein said cryopreserving liquid comprises following composition:
Volumn concentration is the balanced electrolyte solution of 25-70%;
Be the hetastarch of 1-10% by the quality volumn concentration of g/mL;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 5-20%;
Volumn concentration is the clinical grade DMSO of 5-20%;
Volumn concentration is 20% human albumin of 1-50%.
The immune system of umbilical cord mesenchymal stem cells injection scalable patient of the present invention disorder, reduce the release of inflammatory factor, promote the reparation of intestinal and each organ of getting involved, all kinds of toxic and side effects that conventional treatment drug brings are alleviated by short hemopoietic function, thus make patient break away from stomachache, suffer from diarrhoea, have blood in stool, multiple organ involves pathological changes, the puzzlement of all kinds of disease of hematopoietic system that conventional hormone, willow nitrogen sulphur arsenic pyridine medicine bring, thus reduce mortality rate and the disability rate of inflammatory bowel, and significantly improve life in patients.
Wherein, electrolyte solution and human albumin maintain crystalloid osmotic pressure and colloid osmotic pressure respectively, make cell be in a good osmotic pressure environment, are conducive to growth; DMSO is that routine clinical uses 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; The adenosine triphosphate disodium salt magnesium chloride composition comprised in injection, directly can provide energy to the cell after cryopreservation resuscitation, increases the motility rate after cell recovery, maintains the homergy ability of cell; Injection is for after focus in-situ injection or venoclysis, and the primary power when adenosine triphosphate support mescenchymal stem cell comprised in injection is assembled at culprit lesion place, is increased in sufferer place mescenchymal stem cell aggregate amount, accelerates 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 ATP in injection can play and as above acts in repeatedly infusion process.
In the present invention, the volumn concentration of described balanced electrolyte solution in described cryopreserving liquid is 25-70%, such as 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%.
In the present invention, described hetastarch is 1-10% by the quality volumn concentration of g/mL in described cryopreserving liquid, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.
In the present invention, described adenosine triphosphate disodium salt magnesium chloride is 5-20% by the quality volumn concentration of g/mL in described cryopreserving liquid, such as 5%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.
In the present invention, the volumn concentration of described clinical grade DMSO in described cryopreserving liquid is 5-20%, such as 5%, 8%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.
In the present invention, the volumn concentration of described 20% human albumin in described cryopreserving liquid is 1-50%, such as 1%, 5%, 10%, 12%, 15%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 33%, 35%, 38%, 40%, 42%, 44%, 46%, 48% or 50%.
As the preferred technical solution of the present invention, described cryopreserving liquid comprises following composition:
Volumn concentration is the balanced electrolyte solution of 30-60%;
Be the hetastarch of 5-10% by the quality volumn concentration of g/mL;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 10-20%;
Volumn concentration is the clinical grade DMSO of 10-20%;
Volumn concentration is 20% human albumin of 10-40%.
As the preferred technical solution of the present invention, described cryopreserving liquid comprises following composition:
Volumn concentration is the balanced electrolyte solution of 40-50%;
Be the hetastarch of 6% by the quality volumn concentration of g/mL;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 10%;
Volumn concentration is the clinical grade DMSO of 10%;
Volumn concentration is 20% human albumin of 24-34%.
As the preferred technical solution of the present invention, described cryopreserving liquid comprises following composition:
Volumn concentration is the balanced electrolyte solution of 44%;
Be the hetastarch of 6% by the quality volumn concentration of g/mL;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 10%;
Volumn concentration is the clinical grade DMSO of 10%;
Volumn concentration is 20% human albumin of 30%.
According to different sufferer physical condition and to contained material medication taboo, balanced electrolyte solution of the present invention can receive the combination of any one or at least two kinds in ringer's injection, inverted sugar electrolytes injection or 0.9% sodium chloride injection for Bomaili A injection, acetic acid receive ringer's injection, lactic acid.Such as, hyperkalemia, hypernatremia, metabolic complete for the heart, Liver and kidney function or respiratory alkalosis patient or the patient that accepts steroid hormone or thyroliberin treatment will be cautious use of Bomaili A injection; Gerontal patient often has invisible cardiorenal function complete, should be cautious use of acetic acid and receive ringer's injection.
Each constituent content of Bomaili A injection of the present invention is: every 1000mL contains sodium chloride 5.26g, gluconic acid sodium salt 5.02g, sodium acetate 3.68g, potassium chloride 0.37g and magnesium chloride 0.30g.
Acetic acid of the present invention is received each constituent content of ringer's injection and is: containing sodium chloride 6.0g, sodium acetate 3.8g, potassium chloride 0.30g and calcium chloride 0.20g in every 1000mL.
Lactic acid of the present invention is received each constituent content of ringer's injection and is: containing sodium lactate 3.10g, sodium chloride 6.00g, potassium chloride 0.30g and calcium chloride 0.20g in every 1000mL.
Each constituent content of inverted sugar electrolytes injection of the present invention is: 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.
0.9% sodium chloride injection of the present invention can maintain osmotic pressure.
20% human albumin of the present invention is the human blood albumin products according to States Pharmacopoeia specifications, and the concentration of human albumin is 20% (v/v).
Hetastarch of the present invention can be but be not limited to hetastarch 130/0.4, and its chemical name is poly-(oxygen-2-ethoxy) starch 130/0.4, is that clinical practice blood volume expands agent, has good cytoprotection.
Adenosine triphosphate disodium salt magnesium chloride: ribosidoadenine-5 '-triguaiacyl phosphate disodium salt.For the aseptic freeze-dried product of adenosine triphosphate disodium salt magnesium chloride, every bottle contains adenosine triphosphate disodium salt 100mg, magnesium chloride 32mg, is cellular energy tonic, to freeze-stored cell and the equal Yellow Gentian Extract of myocardial cell.
In addition, hetastarch is added in the present invention as buffer agent in injection, adenosine triphosphate disodium salt magnesium chloride provides energy for cell recovery, can make frozen umbilical cord mesenchymal stem cells injection after thawing cell survival rate improve, thus ensure cell viability.
DMSO of the present invention (dimethyl sulfoxide) is clinical grade, as cell cryopreservation protective agent.
Balanced electrolyte solution of the present invention, hetastarch, adenosine triphosphate disodium salt magnesium chloride, clinical grade DMSO and 20% human albumin all can business buy.
In umbilical cord mesenchymal stem cells injection of the present invention, in described umbilical cord mesenchymal stem cells injection, umbilical cord mesenchymal stem cells content is 5 × 10
6-1 × 10
7/ mL, such as 5 × 10
6/ mL, 6 × 10
6/ mL, 7 × 10
6/ mL, 8 × 10
6/ mL, 9 × 10
6/ mL or 1 × 10
7/ mL.
On the other hand, the invention provides the preparation method of described umbilical cord mesenchymal stem cells injection, said method comprising the steps of:
(1) by balanced electrolyte solution, hetastarch, adenosine triphosphate disodium salt magnesium chloride and 20% human albumin mixing, bacterial filter is used to filter, for subsequent use;
(2) umbilical cord mesenchymal stem cells is added in the solution after filtering to step (1);
(3) step (2) gained cell mixture is added in clinical grade DMSO obtain described umbilical cord mesenchymal stem cells injection, preferably, also comprise the step of the umbilical cord mesenchymal stem cells injection obtained being carried out subpackage.
In the preparation method of umbilical cord mesenchymal stem cells injection of the present invention, step (1) described bacterial filter is 0.22 μm of bacterial filter.
In the preparation method of umbilical cord mesenchymal stem cells injection of the present invention, the content of step (2) described umbilical cord mesenchymal stem cells in gained umbilical cord mesenchymal stem cells injection is 5 × 10
6-1 × 10
7/ mL.
Step of the present invention (3) described subpackage can be point be filled to appropriate gauge
in cell cryopreservation bag.
The standard that in the present invention, " umbilical cord mesenchymal stem cells " reference " international cell therapy association (ISCT) " of indication is formulated, has following technical characteristic: under Standard culture conditions, be attached at frosting; Express CD105, CD73, CD90 and HLA-ABC, do not express CD45, CD34, HLA-DR; In vitro can through being induced to differentiate into chondroblast, adipose cell and osteoblast, fresh cultured or the cell survival rate after recovering are more than 85%.
The present invention carries out separation and Culture with reference to prior art to umbilical cord mesenchymal stem cells, and concrete steps are as follows:
Get the fresh umbilicus band of people, clean in D-Hanks liquid, and use Mechanical Method that umbilical cord tissue shearing is become fritter, and the arteries removed wherein, vein blood vessel, the impurity such as the clot of condensation, after further chopping, reuse D-Hanks liquid to clean, be immersed in containing 10% Collagenase after measurement volumes, 5% hyaluronidase, 5% neutral protease, digestion 2 hours is shaken in the premix digestion solution of 80%D-Hanks liquid, screen cloth is used to filter, and clean, be sub-packed in culture bottle or culture dish, add UMSC-CM2 culture medium solution, be placed in CO
2adherent in incubator.Replaced medium after cell attachment, after carried out changing liquid every 2-4 days, cell attachment uses serum-free pancreatin to carry out had digestive transfer culture cultivation after reaching and merging, and the 2-5 that goes down to posterity washs for rear PBS solution, resuspended.
Umbilical cord mesenchymal stem cells after cultivating can directly use, after utilizing normal saline (0.9% sodium chloride injection) resuspended by cell, according to general venous transfusion flow process, cell is added in 0.9% sodium chloride injection, and cell concentration is adjusted to 5 × 10
6-1 × 10
7/ mL, can direct intravenously administrable.But in order to while guarantee cell viability and medical safety and effectiveness, clinical direct infusion is facilitated to use, be convenient to transport and store, liquid nitrogen (-196 DEG C) can be utilized frozen described in the invention provides, and after recovery of thawing direct infusion (use put into preheating in advance 37 DEG C of water-baths carry out recovery of thawing), without the need to again cultivating the cell after recovery of thawing, and directly can be used for treating inflammatory bowel.
On the other hand, the invention provides the application of described umbilical cord mesenchymal stem cells injection in the medicine of preparation treatment inflammatory bowel.
Injection provided by the present invention can be used for the treatment of inflammatory bowel, reduces mortality rate and disability rate, significantly improves life in patients.
Relative to prior art, the present invention has following beneficial effect:
The preparation method of umbilical cord mesenchymal stem cells injection provided by the invention is simple, material is easy to get, obtained umbilical cord mesenchymal stem cells injection is without animal serum, definite ingredients, safety is controlled, and it is effective to possess cell cryopreservation, feature energetic after recovery, long storage periods and the long-distance transport of umbilical cord mesenchymal stem cells preparation can be ensured, this injection can be used in the whole nation and the quality of the pharmaceutical preparations is protected safely on a large scale.Umbilical cord mesenchymal stem cells injection of the present invention integrates frozen and injection, thawing after recovery, without the need to cultivating wherein cell, can be directly used in infusion again to injection.During the umbilical cord mesenchymal stem cells injection for treating mice inflammatory bowel prepared with the present invention, the survival rate of mice is 70%-80%, relative to matched group, significantly improve mouse survival rate, and Mouse Weight recovers very fast, and scoring and pathological score all decrease relative to matched group substantially, therefore, umbilical cord mesenchymal stem cells injection of the present invention is better for the therapeutic effect of inflammatory bowel, and has no side effect.
Accompanying drawing explanation
Fig. 1 is the cellular morphology figure of the umbilical cord mesenchymal stem cells that embodiment 1 obtains, and wherein A figure is the cellular morphology figure after being separated inoculated and cultured 2d, and B is the cellular morphology figure after cell separation inoculated and cultured 5d;
Fig. 2 is the result figure of the immunophenotype of the umbilical cord mesenchymal stem cells utilizing flow cytomery embodiment 1 to obtain;
Fig. 3 is that the umbilical cord mesenchymal stem cells that embodiment 1 obtains passes through induction to osteoblast, lipoblast and Chondrocyte Differentiation figure, and wherein A figure is for being divided into osteoblast, and B figure is for being divided into lipoblast, and C figure is for being divided into chondrocyte;
Fig. 4 is the result figure of the umbilical cord mesenchymal stem cells injection immunophenotype of umbilical cord mesenchymal stem cells after recovery in frozen 2 years utilizing flow cytomery embodiment 2 to prepare;
Fig. 5 be the umbilical cord mesenchymal stem cells injection prepared of embodiment 2 after recovery in frozen 2 years umbilical cord mesenchymal stem cells through the differentiation figure of induction to osteoblast, lipoblast and chondrocyte, wherein A figure is for being divided into osteoblast, B figure is for being divided into lipoblast, and C figure is for being divided into chondrocyte;
Fig. 6 utilizes the growth curve (A) of umbilical cord mesenchymal stem cells injection for treating mice inflammatory bowel process small mouse and change (B) figure of Mouse Weight relative percentage in embodiment 10;
Fig. 7 be by embodiment 10 by treatment after mice dissect after colon state diagram;
Fig. 8 is the optics Electronic Speculum figure after utilizing umbilical cord mesenchymal stem cells injection for treating mice postcolon to organize HE to dye in embodiment 10, and wherein A figure is matched group, and B figure is tail intravenous transplantation group, and C figure is intraperitoneal transplantation treatment group.
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.
embodiment 1
In the present embodiment, carry out separation and Culture to umbilical cord mesenchymal stem cells by the following method, concrete steps are as follows:
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, aids antibody, negative), placed by fresh umbilical cord in the special preservation bottle containing D-Hanks balance liquid, low temperature ice box transports;
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;
It is about 2-5cm segment that umbilical cord tissue after cleaning is carried out mechanical shearing, 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;
Fresh is containing the premix digestion solution of 10% Collagenase (U.S. Sigma), 5% hyaluronidase (U.S. Sigma), 5% neutral protease (U.S. 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;
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 37 DEG C, CO
2cultivate in incubator, use serum-free trypsinization until Growth of Cells after merging state, centrifugal, go down to posterity in subpackage to multiple culture bottle, the 2-5 that goes down to posterity, for rear observation of cell state, uses PBS buffer solution, carries out qualification for subsequent use after trypsinization.
Utilize instrument (Olympus, IX83 inverted microscope) observe in embodiment 1 cellular morphology of umbilical cord mesenchymal stem cells after 2-5 generation of going down to posterity, result as shown in Figure 1, cell cultivation after 5 days cellular morphology become more typical umbilical cord mesenchyma sample, and there is obvious adherent feature, can frosting be attached at.
Utilize flow cytometer (BD, CANTOII) immunophenotype of the umbilical cord mesenchymal stem cells that embodiment 1 obtains is detected, testing result as shown in Figure 2, in Table 1 result is summed up, express CD105, CD73, CD90 and HLA-ABC (expression is higher than 90%) by the known umbilical cord mesenchymal stem cells of result, and do not express CD45, CD34, HLA-DR (expression is lower than 1%).
Table 1
| Traget antibody | Positive rate (%) | Traget antibody | Positive rate (%) |
| CD34 | 0 | CD90 | 99.58 |
| CD44 | 99.48 | CD105 | 94.43 |
| CD45 | 0 | HLA-DR | 0.02 |
| CD73 | 99.72 | HLA-ABC | 96.27 |
Carry out differentiation-inducing to the umbilical cord mesenchymal stem cells that embodiment 1 obtains in vitro, as shown in Figure 3, umbilical cord mesenchymal stem cells can through being induced to differentiate into chondroblast, adipose cell and osteoblast, through alkaline phosphatase staining, (A schemes, chondrocyte), Alizarin red staining (B schemes, adipose cell), oil red " 0 " dyeing (C schemes, osteoblast) all can at Microscopic observation to positive.
The standard that qualification result is formulated with reference to " international cell therapy association (ISCT) ", provable prepared cell possesses mescenchymal stem cell characteristic.
embodiment 2
In the present embodiment, prepare umbilical cord mesenchymal stem cells injection by the following method, infusion pump is containing mescenchymal stem cell and cryopreserving liquid, and wherein cryopreserving liquid comprises following composition:
Volumn concentration is the Bomaili A injection of 44%;
By the hetastarch 130/0.4 that the quality volumn concentration of g/mL is 6%;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 10%;
Volumn concentration is the clinical grade DMSO of 10%;
Volumn concentration is 20% human albumin of 30%.
Be prepared by the following method, specifically comprise the following steps:
(1) Bomaili A injection (Shanghai Baxter Healthcare Ltd.), hetastarch 130/0.4 (together Pharmaceutical), adenosine triphosphate disodium salt magnesium chloride (Shanxi Yabao Pharmaceutical Group Corp.) and 20% human albumin (Shanghai Laishi Blood Product Co., Ltd) are mixed by above-mentioned percent by volume, 0.22 μm of bacterial filter is used to filter, for subsequent use;
(2) add umbilical cord mesenchymal stem cells in the solution after filtering to step (1), controlling its density in final injection is 5 × 10
6-1 × 10
7/ mL;
(3) step (2) gained cell mixture is added in clinical grade DMSO (German WAKCHEMIE), be sub-packed in
namely umbilical cord mesenchymal stem cells injection is obtained in cell cryopreservation bag (German U.S. sky Ni).
The umbilical cord mesenchymal stem cells injection be divided in frozen bag of embodiment 2 preparation is kept sample and carries out a batch detection, test item is that endotoxin detects (gel method), antibacterial culturing, Viral diagnosis, detection of mycoplasma, hepatitis B surface antigen, c-hepatitis antibody, syphilis antibody, aids antibody, finds that above-mentioned testing result is all negative after testing.
Umbilical cord mesenchymal stem cells injection embodiment 2 prepared carries out freezing through Slow-rate freezing instrument, after put into liquid nitrogen and carry out preservation 6 months; Get same cell concentration simultaneously, take conventional cryopreservation liquid (DMSO10%, hyclone FBS10%, UMSC-CM2 culture medium 80%) to carry out programmed cooling and put into Liquid nitrogen storage contrasting for 6 months, checking recovery vigor.
The cell cryopreservation bag of two 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, clamp frozen bag corner with tweezers and slightly adjust 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, carry out cell counting, calculate motility rate.Result is as described in Table 2.
Table 2
| Embodiment 2 | Conventional cryopreservation liquid | |
| Sample 1 | 95.3％ | 96.8％ |
| Sample 2 | 98.6％ | 94.0％ |
| Sample 3 | 96.6％ | 96.1％ |
As can be seen from Table 2, Cell viability after umbilical cord mesenchymal stem cells injection cryopreservation resuscitation prepared by the present invention is consistent with conventional DMSO-hyclone cryopreserving liquid, reach the same effect, and not containing animal sources, definite ingredients is controlled, there is biological safety, illustrate that it can replace traditional cells frozen storing liquid, and can at the rear direct infusion of recovery of thawing.Owing to not adding hetastarch 130/0.4 and adenosine triphosphate disodium salt magnesium chloride and making Cell viability greatly reduce in comparative example 1, this illustrates that these two kinds of compositions have a significant impact for the Cell viability tool after cryopreservation resuscitation, and it can ensure to obtain higher Cell viability.
The motility rate of umbilical cord mesenchymal stem cells injection rear mescenchymal stem cell motility rate of different time points recovery during frozen 2 years prepared by embodiment 2 is investigated, frozen 6 months are chosen in frozen process, 12 months, 18 months and 24 months points carry out recovery and measure Cell viability, concrete operations are: from liquid nitrogen, take out three parts of cell cryopreservation bags according to storage time point is random, carefully put into the water-bath being preheated to 37 DEG C, clamp frozen bag corner with tweezers and slightly adjust 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, Microscopic observation, carry out cell counting, calculate motility rate, result is as shown in table 3.
Table 3
From table 3, the mescenchymal stem cell 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, 4.4% is declined when motility rate was compared with 6 months, but still keep the high motility rate of 91.7%, ensure therapeutic effect, and provide the long-term possibility of preserving and transporting.
Utilize flow cytometer (BD, CANTOII) the umbilical cord mesenchymal stem cells injection of embodiment 2 preparation is detected after frozen 2 years, the wherein immunophenotype of umbilical cord mesenchymal stem cells, testing result as shown in Figure 4, in table 4 result is summed up, after injection is frozen, still CD105 can be expressed by the known umbilical cord mesenchymal stem cells of result, CD73, CD90 and HLA-ABC (expression is higher than 90%), and do not express CD45, CD34, HLA-DR (expression is lower than 1%), acquired results is consistent with the mescenchymal stem cell phenotype of fresh preparation, confirm to preserve mescenchymal stem cell Surface Phenotype without impact for a long time.
Table 4
| 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 |
Umbilical cord mesenchymal stem cells after the umbilical cord mesenchymal stem cells injection recovery in frozen 2 years prepare embodiment 2 carries out differentiation-inducing, the injection of frozen 24 months is selected to carry out recovery of thawing, cultivate according to general cultural method after washing, then carry out differentiation-inducing in vitro, result as shown in Figure 5.Umbilical cord mesenchymal stem cells can through being induced to differentiate into chondroblast, adipose cell and osteoblast, through alkaline phosphatase staining, (A schemes, chondrocyte), (B schemes Alizarin red staining, adipose cell), oil red " 0 " dyeing (C scheme, osteoblast) all can at Microscopic observation to positive, proves the ability of mescenchymal stem cell in injection still maintenance Multidirectional Differentiation.
embodiment 3
In the present embodiment, prepare umbilical cord mesenchymal stem cells injection by the following method, infusion pump is containing mescenchymal stem cell and cryopreserving liquid, and wherein cryopreserving liquid comprises following composition:
Volumn concentration be 40% acetic acid receive ringer's injection;
By the hetastarch 130/0.4 that the quality volumn concentration of g/mL is 6%;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 10%;
Volumn concentration is the clinical grade DMSO of 10%;
Volumn concentration is 20% human albumin of 34%.
Be prepared by the following method, specifically comprise the following steps:
(1) acetic acid being received ringer's injection (Hunan Kangyuan Pharmacy Co., Ltd.), hetastarch 130/0.4, adenosine triphosphate disodium salt magnesium chloride and 20% human albumin mixes by above-mentioned percent by volume, 0.22 μm of bacterial filter is used to filter, for subsequent use;
(2) add umbilical cord mesenchymal stem cells in the solution after filtering to step (1), controlling its density in final injection is 5 × 10
6-1 × 10
7/ mL;
(3) step (2) gained cell mixture is added in clinical grade DMSO, be sub-packed in
namely umbilical cord mesenchymal stem cells injection is obtained in cell cryopreservation bag.
embodiment 4
In the present embodiment, prepare umbilical cord mesenchymal stem cells injection by the following method, infusion pump is containing mescenchymal stem cell and cryopreserving liquid, and wherein cryopreserving liquid comprises the composition of following percent by volume:
Volumn concentration be 50% lactic acid receive ringer's injection;
By the hetastarch 130/0.4 that the quality volumn concentration of g/mL is 6%;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 10%;
Volumn concentration is the clinical grade DMSO of 10%;
Volumn concentration is 20% human albumin of 24%.
Be prepared by the following method, specifically comprise the following steps:
(1) lactic acid being received ringer's injection (Liuan Huayuan Pharmaceutical Co., Ltd.), hetastarch 130/0.4, adenosine triphosphate disodium salt magnesium chloride and 20% human albumin mixes by above-mentioned percent by volume, 0.22 μm of bacterial filter is used to filter, for subsequent use;
(2) add umbilical cord mesenchymal stem cells in the solution after filtering to step (1), controlling its density in final injection is 5 × 10
6-1 × 10
7/ mL;
(3) step (2) gained cell mixture is added in clinical grade DMSO, be sub-packed in
namely umbilical cord mesenchymal stem cells injection is obtained in cell cryopreservation bag.
embodiment 5
In the present embodiment, prepare umbilical cord mesenchymal stem cells injection by the following method, infusion pump is containing mescenchymal stem cell and cryopreserving liquid, and wherein cryopreserving liquid comprises following composition:
Volumn concentration is the inverted sugar electrolytes injection of 30%;
By the hetastarch 130/0.4 that the quality volumn concentration of g/mL is 5%;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 15%;
Volumn concentration is the clinical grade DMSO of 20%;
Volumn concentration is 20% human albumin of 30%.
Be prepared by the following method, specifically comprise the following steps:
(1) inverted sugar electrolytes injection (Yangzijiang Pharmaceutical Group, Shanghai Haini Pharmaceutical Co., Ltd), hetastarch 130/0.4, adenosine triphosphate disodium salt magnesium chloride and 20% human albumin are mixed by above-mentioned percent by volume, 0.22 μm of bacterial filter is used to filter, for subsequent use;
(2) add umbilical cord mesenchymal stem cells in the solution after filtering to step (1), controlling its density in final injection is 5 × 10
6-1 × 10
7/ mL;
(3) step (2) gained cell mixture is added in clinical grade DMSO, be sub-packed in
namely umbilical cord mesenchymal stem cells injection is obtained in cell cryopreservation bag.
embodiment 6
In the present embodiment, prepare umbilical cord mesenchymal stem cells injection by the following method, infusion pump is containing mescenchymal stem cell and cryopreserving liquid, and wherein cryopreserving liquid comprises the composition of following percent by volume:
Volumn concentration is 0.9% sodium chloride injection of 60%;
By the hetastarch 130/0.4 that the quality volumn concentration of g/mL is 10%;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 10%;
Volumn concentration is the clinical grade DMSO of 10%;
Volumn concentration is 20% human albumin of 10%.
Be prepared by the following method, specifically comprise the following steps:
(1) 0.9% sodium chloride injection (Fourth Ring pharmacy), hetastarch 130/0.4, adenosine triphosphate disodium salt magnesium chloride and 20% human albumin are mixed by above-mentioned percent by volume, use 0.22 μm of bacterial filter to filter, for subsequent use;
(2) add umbilical cord mesenchymal stem cells in the solution after filtering to step (1), controlling its density in final injection is 5 × 10
6-1 × 10
7/ mL;
(3) step (2) gained cell mixture is added in clinical grade DMSO, be sub-packed in
namely umbilical cord mesenchymal stem cells injection is obtained in cell cryopreservation bag.
embodiment 7
In the present embodiment, prepare umbilical cord mesenchymal stem cells injection by the following method, infusion pump is containing mescenchymal stem cell and cryopreserving liquid, and wherein cryopreserving liquid comprises following composition:
Volumn concentration is 0.9% sodium chloride injection of 25% and the mixture of Bomaili A injection;
By the hetastarch 130/0.4 that the quality volumn concentration of g/mL is 10%;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 10%;
Volumn concentration is the clinical grade DMSO of 5%;
Volumn concentration is 20% human albumin of 50%.
Be prepared by the following method, specifically comprise the following steps:
(1) mixture (the two volume ratio is 1:1) of 0.9% sodium chloride injection and Bomaili A injection, hetastarch 130/0.4, adenosine triphosphate disodium salt magnesium chloride and 20% human albumin are mixed by above-mentioned percent by volume, 0.22 μm of bacterial filter is used to filter, for subsequent use;
(2) add umbilical cord mesenchymal stem cells in the solution after filtering to step (1), controlling its density in final injection is 5 × 10
6-1 × 10
7/ mL;
(3) step (2) gained cell mixture is added in clinical grade DMSO, be sub-packed in
namely umbilical cord mesenchymal stem cells injection is obtained in cell cryopreservation bag.
embodiment 8
In the present embodiment, prepare umbilical cord mesenchymal stem cells injection by the following method, infusion pump is containing mescenchymal stem cell and cryopreserving liquid, and wherein cryopreserving liquid comprises following composition:
Volumn concentration is 0.9% sodium chloride injection of 70% and the mixture of Bomaili A injection;
By the hetastarch 130/0.4 that the quality volumn concentration of g/mL is 1%;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 5%;
Volumn concentration is the clinical grade DMSO of 20%;
Volumn concentration is 20% human albumin of 4%.
Be prepared by the following method, specifically comprise the following steps:
(1) mixture (the two volume ratio is 1:1) of 0.9% sodium chloride injection and Bomaili A injection, hetastarch 130/0.4, adenosine triphosphate disodium salt magnesium chloride and 20% human albumin are mixed by above-mentioned percent by volume, 0.22 μm of bacterial filter is used to filter, for subsequent use;
(2) add umbilical cord mesenchymal stem cells in the solution after filtering to step (1), controlling its density in final injection is 5 × 10
6-1 × 10
7/ mL;
(3) step (2) gained cell mixture is added in clinical grade DMSO, be sub-packed in
namely umbilical cord mesenchymal stem cells injection is obtained in cell cryopreservation bag.
embodiment 9
In the present embodiment, prepare umbilical cord mesenchymal stem cells injection by the following method, infusion pump is containing mescenchymal stem cell and cryopreserving liquid, and wherein cryopreserving liquid comprises following composition:
Volumn concentration is 0.9% chloride injection of 70%, the mixture of liquid Bomaili A injection and inverted sugar electrolytes injection;
By the hetastarch 130/0.4 that the quality volumn concentration of g/mL is 4%;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 20%;
Volumn concentration is the clinical grade DMSO of 5%;
Volumn concentration is 20% human albumin of 1%.
Be prepared by the following method, specifically comprise the following steps:
(1) mixture (three's volume ratio is 1:1:1) of 0.9% chloride injection, liquid Bomaili A injection and inverted sugar electrolytes injection, hetastarch 130/0.4, adenosine triphosphate disodium salt magnesium chloride and 20% human albumin are mixed by above-mentioned percent by volume, 0.22 μm of bacterial filter is used to filter, for subsequent use;
(2) add umbilical cord mesenchymal stem cells in the solution after filtering to step (1), controlling its density in final injection is 5 × 10
6-1 × 10
7/ mL;
(3) step (2) gained cell mixture is added in clinical grade DMSO, be sub-packed in
namely umbilical cord mesenchymal stem cells injection is obtained in cell cryopreservation bag.
embodiment 10
Safety, effectiveness that the umbilical cord mesenchymal stem cells injection prepared embodiment 2 is in the present embodiment treated inflammatory bowel animal model are studied, and concrete grammar is as follows:
Select the male BALB/c mouse of SPF rank, body weight 18-22g.Select 2,4,6-trinitro-benzene-sulfonic acid (TNBS) 5% aqueous solution, the acetone-mixed with olive oil solution of 4:1 proportions, the two is formulated as sensitization of skin liquid according to 4:1 ratio (acetone-mixed with olive oil liquid: TNBS); TNBS is become TNBS enema solution with dehydrated alcohol according to 1:1 proportions.
Mouse back is cut off the hair that 1.5cm is square, baring skin, normally raise after every mice gives 150 μ L sensitization of skin liquid, after 7d, use 150mg/kgTNBS coloclysis.Stroking mouse back stimulates far-end stool emptying; After lumbar injection pentobarbital sodium (30mg/kg) anesthesia, hang position by the feet, diameter 0.2mm polyethylene tube is inserted colon 4cm place, slowly pours into enema solution, put back in cage and normally raise.Observe mice stool, the mental status etc., record Mouse Weight change every day.Put to death after 7d, get colon and substantially mark, and get damaged part specimens paraffin embedding slices, after HE (hematoxylin-eosin) dyeing, carry out pathological score.3 groups are divided into: matched group (n=8), 24h tail venoclysis PBS after coloclysis after the modeling of mice TNBS coloclysis; Tail intravenous transplantation group (n=11), 24h after coloclysis, the umbilical cord mesenchymal stem cells injection utilizing embodiment 2 to prepare and 0.9% sodium chloride injection carry out tail vein mixing infusion, and the quantity of the mescenchymal stem cell of every mice infusion is 2 × 10
5; Intraperitoneal transplantation treatment group (n=10), 24h after coloclysis, carries out abdominal cavity infusion after the umbilical cord mesenchymal stem cells injection utilizing embodiment 2 to prepare mixes with 0.9% sodium chloride injection, and the quantity of the mescenchymal stem cell of every mice infusion is 2 × 10
5.
The growing state of each group of mice and Mouse Weight are monitored, result as shown in Figure 6, can find out, after abdominal cavity arteries and veins infusion umbilical cord mesenchymal stem cells injection, the survival rate of mice is more than 80%, after tail venoclysis umbilical cord mesenchymal stem cells injection, the survival rate of mice is more than 70%, relative to matched group, all can improve animal survival rate, abdominal cavity infusion is better than tail intravenous transplantation group improving in mouse survival rate.After umbilical cord mesenchymal stem cells injection is transplanted, tail intravenous transplantation group body weight is slowly gone up; Intraperitoneal transplantation treatment group body weight after the transfer 2d drops to minimum, then slowly gos up, and experimental endpoints weight recovery and tail intravenous transplantation group remain basically stable; Control group mice body weight drops to minimum in 2d body weight, then maintains a stable level.
5d after transplanting, takes out mice total colectomy, observes and mark under anatomic microscope.As shown in Figure 7, control group mice colon all visible ulcer companion hyperemia, edema; Tail intravenous transplantation group mice recovers better, only have individual animal to have ulcer, and pathological changes to be less; Intraperitoneal transplantation treatment group animal colon ulcer alleviates, and extent of disease reduces.Appraisal result is shown in Table 5 substantially, and result all can play repairing effect to ulcer after showing UCMSC tail vein or intraperitoneal transplantation.
Table 5
| Group | Substantially mark | Pathological score |
| Matched group | 3.33±1.22 | 7.78±2.68 |
| Tail vein transplantation group | 1±1.41 | 3.11±2.67 |
| Intraperitoneal transplantation group | 1.57±1.51 | 5.14±1.86 |
Standards of grading are as follows: standards of grading substantially: [0 point: roughly normal; 1 point: without ulcer, contrafluxion; 2 points: visible ulcer, but without hyperemia; 3 points: only 1 place's ulcer and inflammation; The ulcer at 4 points: 2 places or more place and inflammation; 5 points: ulcer is longer than 2cm; 6 ~ 10 points: the ulcer being greater than 2cm, often increase the many meters of 1cm 1 point].Histopathological scores standard: [edema: 0 or 1 (being with or without); Ulcer: 0 or 1 (being with or without); Cellular infiltration: 0,1,2 or 3 (nothing, slightly, severe); Muscle layer thickens: 0,1,2 or 3 (nothing, slightly, severe); Angiogenesis: 0 or 1 (being with or without); Mucosa structure destroys: 0 or 1 (being with or without); Mucosa structure is lost: 0,1,2 or 3 (nothing, slightly, severe); Add up to total score].
Cut into slices after colon paraffin embedding, HE dyes, light microscopic (Olympus; IX83 inverted microscope) under observe, as shown in Figure 8, find that control group mice colon intestinal wall thickens, mucous layer extensive necrosis, a large amount of lymphocytic infiltration holostrome; Tail intravenous transplantation group and intraperitoneal transplantation treatment group animal some only see intestinal wall and slightly thicken, but mucosa is complete, and some only sees slight defect at mucous layer, and lymphocytic infiltration comparatively matched group reduces; Minority mice is visible ulcer still, and a large amount of lymphocytic infiltration also generates with new vessels.Compared with matched group, the colon pathological score of 2 kinds of transplantation treatment modes all significantly declines.
Therefore, when the umbilical cord mesenchymal stem cells injection utilizing the present invention to prepare is treated inflammatory bowel, no matter take venoclysis or abdominal cavity infusion, umbilical cord mesenchymal stem cells all plays therapeutic effect to inflammatory bowel, and compared with matched group, the weight of animals recovers very fast, mortality rate reduces, endo enteritis alleviates, and cardinal principle and pathological score all decrease, and do not observe toxic and side effects on animal model.
Applicant states, the present invention illustrates umbilical cord mesenchymal stem cells injection of the present invention and its preparation method and application by above-described embodiment, but the present invention is not limited to above-described embodiment, does not namely mean that the present invention must rely on above-described embodiment 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 (10)
1. a umbilical cord mesenchymal stem cells injection, is characterized in that, described infusion pump is containing mescenchymal stem cell and cryopreserving liquid, and wherein said cryopreserving liquid comprises following composition:
Volumn concentration is the balanced electrolyte solution of 25-70%;
Be the hetastarch of 1-10% by the quality volumn concentration of g/mL;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 5-20%;
Volumn concentration is the clinical grade DMSO of 5-20%;
Volumn concentration is 20% human albumin of 1-50%.
2. umbilical cord mesenchymal stem cells injection according to claim 1, is characterized in that, described cryopreserving liquid comprises following composition:
Volumn concentration is the balanced electrolyte solution of 30-60%;
Be the hetastarch of 5-10% by the quality volumn concentration of g/mL;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 10-20%;
Volumn concentration is the clinical grade DMSO of 10-20%;
Volumn concentration is 20% human albumin of 10-40%.
3. umbilical cord mesenchymal stem cells injection according to claim 1 and 2, is characterized in that, described cryopreserving liquid comprises following composition:
Volumn concentration is the balanced electrolyte solution of 40-50%;
Be the hetastarch of 6% by the quality volumn concentration of g/mL;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 10%;
Volumn concentration is the clinical grade DMSO of 10%;
Volumn concentration is 20% human albumin of 24-34%.
4. the umbilical cord mesenchymal stem cells injection according to any one of claim 1-3, is characterized in that, described cryopreserving liquid comprises following composition:
Volumn concentration is the balanced electrolyte solution of 44%;
Be the hetastarch of 6% by the quality volumn concentration of g/mL;
By the adenosine triphosphate disodium salt magnesium chloride that the quality volumn concentration of g/mL is 10%;
Volumn concentration is the clinical grade DMSO of 10%;
Volumn concentration is 20% human albumin of 30%.
5. the umbilical cord mesenchymal stem cells injection according to any one of claim 1-4, it is characterized in that, described balanced electrolyte solution is Bomaili A injection, ringer's injection received by acetic acid, lactic acid receives ringer's injection, the combination of any one or at least two kinds in inverted sugar electrolytes injection or 0.9% sodium chloride injection.
6. the umbilical cord mesenchymal stem cells injection according to any one of claim 1-5, is characterized in that, in described umbilical cord mesenchymal stem cells injection, umbilical cord mesenchymal stem cells content is 5 × 10
6-1 × 10
7/ mL.
7. the preparation method of the umbilical cord mesenchymal stem cells injection according to any one of claim 1-6, is characterized in that, said method comprising the steps of:
(1) by balanced electrolyte solution, hetastarch, adenosine triphosphate disodium salt magnesium chloride and 20% human albumin mixing, bacterial filter is used to filter, for subsequent use;
(2) umbilical cord mesenchymal stem cells is added in the solution after filtering to step (1);
(3) step (2) gained cell mixture is added in clinical grade DMSO obtain described umbilical cord mesenchymal stem cells injection, preferably, also comprise the step of the umbilical cord mesenchymal stem cells injection obtained being carried out subpackage.
8. preparation method according to claim 7, is characterized in that, step (1) described bacterial filter is 0.22 μm of bacterial filter.
9. the preparation method according to claim 7 or 8, is characterized in that, the content of step (2) described umbilical cord mesenchymal stem cells in gained umbilical cord mesenchymal stem cells injection is 5 × 10
6-1 × 10
7/ mL.
10. the application of the umbilical cord mesenchymal stem cells injection according to any one of claim 1-6 in the medicine of preparation treatment inflammatory bowel.
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