CN103271943A - Application of stem cell in preparation of preparation used for treating parkinson disease - Google Patents
Application of stem cell in preparation of preparation used for treating parkinson disease Download PDFInfo
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Abstract
The invention provides application of an umbilical cord mesenchymal stem cell in preparation of a preparation used for treating parkinson disease, and also provides a method for preparing a umbilical cord mesenchymal stem cell for a preparation used for treating parkinson disease, and a preparation composition with the stem cell. The prepared umbilical cord mesenchymal stem cell has the advantages of low immunoreactions risk, high activity, low residues, low preparation cost, and the like.
Description
Technical field
The present invention relates to the formulation art of stem cell preparation treatment disease.Concrete, the invention provides the purposes of a kind of umbilical cord mesenchymal stem cells in the Parkinsonian preparation of preparation treatment.
Background technology
Parkinson disease (Parkinson's Disease PD), or is called parkinson, be a kind of be the neurodegenerative diseases of feature with the forfeiture of carrying out property of striatum dopaminergic neuron function.James Parkinson in 1817 described parkinson (Parkinson's Disease, PD) in two self-contradictory symptoms-tremble and benumbing, myotonia is the third-largest symptom.PD is the common impaired disease of ganglion basal, and dopaminergic neuron degeneration in black substance in the ganglion basal-striatum system, forfeiture cause dopamine level to descend, the acetylcholine systemic-function is hyperfunction, subthalamic nuclei and the pallidum medial part is overexcited and muscular hypertonia is the main pathology physiological Foundations of PD dyskinesia.In addition, a large amount of clinical and animal experiment study discoveries, the unusual β ripple that occurs in the ganglion basal is also closely related with the muscular tension of PD.
The parkinsonian dyskinesia is being copied by various animal models and is being goed deep into Mechanism Study.In behavioristics's research of PD, its animal model comprises the chemical lesion model, mechanical damage model and gene genetic model.The chemical lesion model comprises with 6-hydroxy dopamine, 1-methyl-4-phenyl-1,2,4, and for example primates or rodent are handled to animal for 6-tetrahydropyridine (MPTP), N,N'-dimethyl-.gamma..gamma.'-dipyridylium, rotenone.The animal model that 6-hydroxy dopamine (6-OHDA) mediation is set up is one of modal parkinson disease model.The animal model that the MPTP mediation is set up is another kind of common parkinson disease model, can simulate Parkinsonian clinical symptoms and pathology characteristics well.Ataxic gait and posture obstacle are parkinsonian's critical function obstacles, mainly show as: forward lean, elbow, waist, knee joint flexing; Stride is the random little step gait for a short time during walking, and walking speed is slow, and walking rhythm moderate reduces; Stride is more and more littler in the walking process, and walking speed is accelerated simultaneously, presents the gait that almost will run and is called festination; Start and during in the walking of narrow place because fearing dare not take a step to be called to freeze gait.According to the Hoehn-Yahr grade classification, parkinsonian's gait change procedure is divided into three phases: 1.. and early stage symptom is that bradykinesia and lower limb exercise amplitude reduce, and produces the typical gait of random little step.2.. along with further developing of the state of an illness, the patient significant gait occurs and freezes.3.. last, phenomenon such as fall postural reflex forfeiture, disequilibrium appear even in the patient.The behavior observation of parkinson animal model is become the important means of checking parkinson animal model and medicine.
Be applied to detect chemical induction, for example behavioristics's detection method mainly contains in the MPTP PD mouse model of inducing: the experiment of walking platform (Catwalk) animal gait, spacious experiment, roller bearing experiment, swimming test, dimpling experiment, pole-climbing experiment, hang and test and holding rod balance test etc.
Parkinson disease still do not have specific effective Therapeutic Method at present.Traditional chemicals can not be treated parkinson disease completely effectively.In addition, chemicals has been found to have certain side effect.
Stem cell is considered to treat the potential method of neurodegenerative diseases.Stem cell (stem cell) is the pluripotent cell that has self renewal and can be differentiated to form more than one cell types, and this potential makes it that prospect very widely be arranged aspect clinical practice.Embryonic stem cell (embryonic stem cell, ESCs are called for short ES or EK cell) is the class cell that body early embryo or original gonad kind are separated, and has the characteristic of In vitro culture infinite multiplication, self renewal and multidirectional differentiation.No matter in external still internal milieu, the ES cell can both be induced to differentiate into the nearly all cell type of body.Yet embryonic stem cell research is because ethical problem is a field that has much dispute always, and its source is subjected to the restriction of ethics, morals, the comparatively difficulty of drawing materials.
Adult stem cell refers to be present in the undifferentiated cell in the differentiated tissue, has self replication and differentiation produces the ability that immature cell is organized in one or more filial generations, can be distributed in adult tissue or the organ.Under given conditions, adult stem cell or produce new stem cell perhaps by the certain procedure differentiation, forms new functioning cell, thus the dynamic equilibrium that makes tissue and organ maintenance grow and fail.
Some researchs have confirmed to substitute after the differentiation of stem cells that to repair the cell that damages be feasible with the treatment nervous system disease.Fu YS etc. were at (Stem cells in 2006,2006,24 (1): 115-124) report, in the striatum with the PD rat model model of not inducing and pass through people's umbilical cord stem cell transplantation of inducing to bring out to 6-hydroxyl DOPA, the tyrosine hydroxylase positive cell of table of discovery intelligent specificity nuclear antigen can be survived at transplantation site and be reached more than April, can be to head, the about 1.4mm of tail both sides migration of transplantation site, the rat that amfetamine the brings out behavior of turn-taking also significantly improves.The tyrosine hydroxylase positive cell of this prompter's umbilical cord source of human stem cell can be used as the Parkinsonian a kind of stem cell for the treatment of.
This area still needs to obtain to control the key molecule of stem cells hyperplasia, avoid controlling the undue growth of neural stem cell, and the function integration that how to realize noble cells and already present cell better, thereby can reach for example Parkinsonian stem cell medicine for the treatment of neurodegenerative diseases.
Summary of the invention
The invention provides the application of umbilical cord mesenchymal stem cells in the Parkinsonian preparation of preparation treatment.It is a kind of with the Parkinsonian method of stem-cell therapy that the present invention also provides.In yet another aspect, the invention provides the method that preparation can be used in the umbilical cord mesenchymal stem cells of the Parkinsonian preparation for the treatment of.The umbilical cord mesenchymal stem cells that utilizes the present invention to prepare has low immunoreation risk, active high, residual advantage such as low, and preparation cost is cheap.Umbilical cord mesenchymal stem cells provided by the invention can be treated parkinson disease effectively.
The invention provides the application of a kind of umbilical cord mesenchymal stem cells in the Parkinsonian preparation of preparation treatment.
In one aspect of the invention, the described umbilical cord mesenchymal stem cells in the above-mentioned application prepares by the following method:
Step 1: obtain umbilical cord China Tong Shi glue; For example, can with the ethanol disinfection sterilization, reject the blood vessel of umbilical cord by the umbilical cord that obtains with the sodium chloride flushing, take out magnificent Tong Shi glue then, use the sodium chloride washing colloids;
Step 2: piece of tissue preparation and inoculation, wherein described colloid is sheared into about 1～4mm
3The tissue homogenate piece, be about 0.4～0.7g/ml with serum-free medium standardize solution tissue homogenate concentration, be inoculated in the culture bottle after the piping and druming homogenate evenly, add serum-free medium and cultivate;
Step 3:P0 is for cell culture, wherein above-mentioned culture bottle is positioned over CO2 gas incubator and cultivates, and is cultured to the 5th～7 day and carries out full dose to change liquid; Be cultured to the 10th～13 day and carry out half amount and change liquid; Be cultured to the 14th～18 day, the cell fusion degree reaches at 70%～80% o'clock, obtains P0 for cell with digestive enzyme digestion results;
Step 4:P1 is for cell culture, wherein with described P0 for cell according to about 5 * 10
3Individual/cm
2Density is inoculated in the culture bottle, is positioned over CO2 gas incubator and cultivates, and cell culture to the 2～4 days after the cell fusion degree reaches 80%～90%, obtains P1 for cell with digestive enzyme digestion results;
Step 5:P2 is for cell culture, wherein with described P1 for cell according to about 5 * 10
3Individual/cm
2Density is inoculated in the culture bottle, and be positioned over CO2 gas incubator and cultivate, cell culture to the 2～4 days, the cell fusion degree reaches 85%～90%, obtains P2 for cell with digestive enzyme digestion results;
Step 6:P3 is for cell culture, wherein with described P2 for cell inoculation in culture bottle, be positioned over CO2 gas incubator and be cultured to the cell fusion degree and reach〉after 95%, obtain P3 for cell with digestive enzyme digestion results;
Step 7:P4 is for cell culture, wherein with described P3 for cell according to about 5 * 10
3Individual/cm
2Density is inoculated in the culture bottle, is positioned over CO2 gas incubator and cultivates, and cell culture to the 2～4 days after the cell fusion degree reaches 85%～90%, obtains P4 for cell with digestive enzyme digestion results.This P4 is for namely being used for aforementioned umbilical cord mesenchymal stem cells for the treatment of the application of Parkinsonian preparation in preparation.
Of the present invention aspect one of them, the step 5 of above-mentioned preparation method comprises that also the described P2 that digestive enzyme digestion results are obtained is for cell cryopreservation, wherein with described P2 for cell as requested frozen density be suspended in the cryopreserving liquid, be positioned in the procedural cooling instrument be down to below-80 ℃ frozen.Aspect one of them, described frozen density is 1～5 * 10 of the present invention
7/ mL.Cryopreserving liquid can comprise basal liquid, permeability cryoprotective agent and impermeability cryoprotective agent.Described basal liquid generally includes culture fluid, phosphate buffer PBS, normal saline etc.Described permeability cryoprotective agent comprises one or more the combination in dimethyl sulfoxide, ethylene glycol, propylene glycol, the glycerol.Described impermeability cryoprotective agent comprises one or more the combination in sucrose, mannitol, the trehalose.
Of the present invention aspect one of them, the step 5 of above-mentioned preparation method also comprises frozen P2 for cell recovery, wherein with described frozen P2 for cell in the about 40.0 ℃ of following water-baths of temperature, rock rewarming fast, cell suspension is by solid-state when becoming liquid state, be seeded in the Tissue Culture Flask, recovery is namely finished.
Of the present invention aspect one of them, in the step 3 of above-mentioned preparation method, when cell culture to the in the time of 14～16 days, if the cell fusion degree does not reach 70%～80%, carry out once half amount again and change liquid.
Aspect one of them, wherein in the cell culture of the step 3-7 of above-mentioned preparation method, all adopt serum-free medium to carry out cell culture of the present invention.In preparation method of the present invention, preferably adopt serum-free medium that P0, P1, P2, P3, P4 are cultivated for cell.Aspect one of them, P0, P1, P2, P3, P4 adopt serum-free medium for the cultivation of cell of the present invention.
In the present invention, serum-free medium refers to not contain the cell culture medium of serum.Can adopt various serum-free medium for cell culture known in the art.Commercially available serum-free medium comprises, for example the Ultraculture of LONZA company
TM, GIBCO company
The STEMPRO MSC SFM of MSC SFM XenoFree, Invitrogen company etc.Be not bound by any theory, the inventor thinks, one of them important advantage of the umbilical cord mesenchymal stem cells that the present invention prepares is a little less than the immunogenicity, not the induction of immunity rejection; Characteristic with similar " immunity is exempted " can suppress the alloimmunity reaction, the anti-host disease of inhibition of transplant.For example parkinson disease are significant to effective treatment neurodegenerative diseases for this.
Aspect one of them, digestive enzyme described in the above-mentioned preparation method is trypsin of the present invention.
Aspect one of them, also comprise step 8 of the present invention in the above-mentioned preparation method, wherein the umbilical cord mesenchymal stem cells to described P4 generation carries out surface antigen check and screening.Of the present invention aspect one of them, screening surface antigen CD90, CD29, CD73, CD105 express the umbilical cord mesenchymal stem cells greater than 99%.Of the present invention aspect one of them, screening surface antigen CD45, CD34, CD19, CD14, HLA-DR express the umbilical cord mesenchymal stem cells less than 2%.Wherein aspect another, screening surface antigen CD90, CD29, CD73, CD105 express greater than 99% of the present invention, and surface antigen CD45, CD34, CD19, CD14, HLA-DR expression are all less than 2% umbilical cord mesenchymal stem cells.
Of the present invention aspect one of them, the step 8 in the above-mentioned preparation method is that the umbilical cord mesenchymal stem cells to described P4 generation carries out the skeletonization conversion ratio and becomes the fat conversion ratio to detect and screening., be filtered into the fat conversion ratio and be the umbilical cord mesenchymal stem cells of about 20-30% aspect one of them of the present invention.Of the present invention aspect one of them, the skeletonization conversion ratio that screens described umbilical cord mesenchymal stem cells is greater than 70% umbilical cord mesenchymal stem cells.Wherein aspect another, being filtered into the fat conversion ratio is about 20-30%, and the skeletonization conversion ratio is greater than 70% umbilical cord mesenchymal stem cells of the present invention.
Of the present invention aspect one of them, the step 8 in the above-mentioned preparation method is that the umbilical cord mesenchymal stem cells to described P4 generation carries out surface antigen check and screening, and carries out the skeletonization conversion ratio and becomes the detection of fat conversion ratio and screen.Of the present invention wherein aspect another, screening surface antigen CD90, CD29, CD73, CD105 express greater than 99%, and surface antigen CD45, CD34, CD19, CD14, HLA-DR express less than 2%, and the skeletonization conversion ratio is greater than 70%, and becomes the fat conversion ratio to be the umbilical cord mesenchymal stem cells of about 20-30%.
Aspect one of them, in the application of umbilical cord mesenchymal stem cells of the present invention in the Parkinsonian preparation of preparation treatment, the amount of the umbilical cord mesenchymal stem cells that described preparation contains is about 1 * 10 of the present invention
6Cell/kg～9 * 10
6Cell.
Aspect one of them, in the application of umbilical cord mesenchymal stem cells of the present invention in the Parkinsonian preparation of preparation treatment, the dosage of the umbilical cord mesenchymal stem cells that described preparation contains is about 1 * 10 of the present invention
6Cell/kg～9 * 10
6Cell/kg.Described dosage refers to give the patient of needs treatment of every kilogram of (kg) weight or the number of other individual stem cell.
The present invention also provides a kind of method for the preparation of the umbilical cord mesenchymal stem cells in the Parkinsonian preparation for the treatment of, comprising:
Step 1: obtain umbilical cord China Tong Shi glue; For example, can with the ethanol disinfection sterilization, reject the blood vessel of umbilical cord by the umbilical cord that obtains with the sodium chloride flushing, take out magnificent Tong Shi glue then, use the sodium chloride washing colloids;
Step 2: piece of tissue preparation and inoculation, wherein described colloid is sheared into about 1～4mm
3The tissue homogenate piece, be about 0.4～0.7g/ml with serum-free medium standardize solution tissue homogenate concentration, be inoculated in the culture bottle after the piping and druming homogenate evenly, add serum-free medium and cultivate;
Step 3:P0 is for cell culture, wherein above-mentioned culture bottle is positioned over CO2 gas incubator and cultivates, and is cultured to the 5th～7 day and carries out full dose to change liquid; Be cultured to the 10th～13 day and carry out half amount and change liquid; Be cultured to the 14th～18 day, the cell fusion degree reaches at 70%～80% o'clock, obtains P0 for cell with digestive enzyme digestion results;
Step 4:P1 is for cell culture, wherein with described P0 for cell according to about 5 * 10
3Individual/cm
2Density is inoculated in the culture bottle, is positioned over CO2 gas incubator and cultivates, and cell culture to the 2～4 days after the cell fusion degree reaches 80%～90%, obtains P1 for cell with digestive enzyme digestion results;
Step 5:P2 is for cell culture, wherein with described P1 for cell according to about 5 * 10
3Individual/cm
2Density is inoculated in the culture bottle, and be positioned over CO2 gas incubator and cultivate, cell culture to the 2～4 days, the cell fusion degree reaches 85%～90%, obtains P2 for cell with digestive enzyme digestion results;
Step 6:P3 is for cell culture, wherein with described P2 for cell inoculation in culture bottle, be positioned over CO2 gas incubator and be cultured to the cell fusion degree and reach〉after 95%, obtain P3 for cell with digestive enzyme digestion results;
Step 7:P4 is for cell culture, wherein with described P3 for cell according to about 5 * 10
3Individual/cm
2Density is inoculated in the culture bottle, is positioned over CO2 gas incubator and cultivates, and cell culture to the 2～4 days after the cell fusion degree reaches 85%～90%, obtains P4 for cell with digestive enzyme digestion results.This P4 is for namely being used for aforementioned umbilical cord mesenchymal stem cells for the treatment of the application of Parkinsonian preparation in preparation.
Of the present invention aspect one of them, the step 5 of above-mentioned preparation method comprises that also the described P2 that digestive enzyme digestion results are obtained is for cell cryopreservation, wherein with described P2 for cell as requested frozen density be suspended in the cryopreserving liquid, be positioned in the procedural cooling instrument be down to below-80 ℃ frozen.Aspect one of them, described frozen density is 1～5 * 10 of the present invention
7/ mL.
Of the present invention aspect one of them, the step 5 of above-mentioned preparation method also comprises frozen P2 for cell recovery, wherein with described frozen P2 for cell in the about 40.0 ℃ of following water-baths of temperature, rock rewarming fast, cell suspension is by solid-state when becoming liquid state, be seeded in the Tissue Culture Flask, recovery is namely finished.
Of the present invention aspect one of them, in the step 3 of above-mentioned preparation method, when cell culture to the in the time of 14～16 days, if the cell fusion degree does not reach 70%～80%, carry out once half amount again and change liquid.
Aspect one of them, wherein in the cell culture of the step 3-7 of above-mentioned preparation method, all adopt serum-free medium to carry out cell culture of the present invention.In preparation method of the present invention, preferably adopt serum-free medium that P0, P1, P2, P3, P4 are cultivated for cell.Aspect one of them, P0, P1, P2, P3, P4 adopt serum-free medium for the cultivation of cell of the present invention.
Aspect one of them, digestive enzyme described in the above-mentioned preparation method is trypsin of the present invention.
Aspect one of them, also comprise step 8 of the present invention in the above-mentioned preparation method, wherein the umbilical cord mesenchymal stem cells to described P4 generation carries out surface antigen check and screening.Wherein aspect another, screening surface antigen CD90, CD29, CD73, CD105 express greater than 99% of the present invention, and surface antigen CD45, CD34, CD19, CD14, HLA-DR expression are all less than 2% umbilical cord mesenchymal stem cells.
Of the present invention aspect one of them, the step 8 in the above-mentioned preparation method is that the umbilical cord mesenchymal stem cells to described P4 generation carries out the skeletonization conversion ratio and becomes the fat conversion ratio to detect and screening.Wherein aspect another, being filtered into the fat conversion ratio is about 20-30%, and the skeletonization conversion ratio is greater than 70% umbilical cord mesenchymal stem cells of the present invention.
Of the present invention aspect one of them, the step 8 in the above-mentioned preparation method is that the umbilical cord mesenchymal stem cells to described P4 generation carries out surface antigen check and screening, and carries out the skeletonization conversion ratio and becomes the detection of fat conversion ratio and screen.Of the present invention wherein aspect another, screening surface antigen CD90, CD29, CD73, CD105 express greater than 99%, and surface antigen CD45, CD34, CD19, CD14, HLA-DR express less than 2%, and the skeletonization conversion ratio is greater than 70%, and becomes the fat conversion ratio to be the umbilical cord mesenchymal stem cells of about 20-30%.
The present invention also provides a kind of Parkinsonian preparation compositions that is used for the treatment of.Above-mentioned preparation compositions contains umbilical cord mesenchymal stem cells.Wherein said umbilical cord mesenchymal stem cells is by adopting aforesaid method preparation.The dosage of the umbilical cord mesenchymal stem cells that described preparation contains can be about 1 * 10
6Cell/kg～9 * 10
6Cell/kg.Described dosage refers to give the patient of needs treatment of every kilogram of (kg) weight or the number of other individual stem cell.
Be used for the treatment of in the Parkinsonian preparation compositions of the present invention, surface antigen CD90, the CD29 of described umbilical cord mesenchymal stem cells, CD73, CD105 express greater than 99%.Aspect one of them, surface antigen CD45, the CD34 of described umbilical cord mesenchymal stem cells, CD19, CD14, HLA-DR express less than 2% of the present invention.Wherein aspect another, surface antigen CD90, the CD29 of described umbilical cord mesenchymal stem cells, CD73, CD105 express greater than 99% of the present invention, and surface antigen CD45, CD34, CD19, CD14, HLA-DR express less than 2%.
, be used for the treatment of in the Parkinsonian preparation compositions of the present invention aspect one of them of the present invention, described umbilical cord mesenchymal stem cells has skeletonization and becomes the fat differentiation capability.Aspect one of them, the one-tenth fat conversion ratio of described umbilical cord mesenchymal stem cells is about 20-30% of the present invention.Aspect one of them, the skeletonization conversion ratio of described umbilical cord mesenchymal stem cells is greater than 70% of the present invention.Wherein aspect another, the one-tenth fat conversion ratio of described umbilical cord mesenchymal stem cells is about 20-30%, and the skeletonization conversion ratio is greater than 70% of the present invention.
Wherein aspect another, also comprise cell culture medium of the present invention in the above-mentioned preparation compositions, be preferably serum-free cell culture medium.
The inventor is surprised to find that the umbilical cord mesenchymal stem cells that utilizes the present invention to prepare has the advantage that is different from prior art: a little less than the immunogenicity, and induction of immunity rejection not; Characteristic with similar " immunity is exempted " can suppress the alloimmunity reaction, the anti-host disease of inhibition of transplant (GVHD).The method that the present invention prepares umbilical cord mesenchymal stem cells is simple, and the cell viability that obtains is very strong, easily a large amount of amplifications, and this may to have a longer telomere relevant with it.For example, according to above-mentioned technology of the present invention, per 1 bottle of P0 is for cell (3 * 10
3), be passaged to P4 generation, can obtain 625 bottles, 3 * 10
6Cell/bottle.This is the excellent results that cell culture system of the present invention, culture process can be obtained.The more important thing is that the inventor is surprised to find that, with the umbilical cord mesenchymal stem cells of method preparation of the present invention, can treat parkinson disease effectively.This is the character with the present invention preparation and the umbilical cord mesenchymal stem cells that adopts, the positive index of particular surface labelling that comprises umbilical cord mesenchymal stem cells and negative index with and the advantage that becomes fat Osteoblast Differentiation ability etc. to bring.In undocumented laboratory and clinical experiment, find, the growth stage of umbilical cord mesenchymal stem cells may have critical impact to treatment, does not reach the positive index of particular surface labelling and negative index and/or have specificly to become the stem cell of fat Osteoblast Differentiation ability can not obtain satisfied curative effect.
In this application, the accessible range of error of statement " pact " expression those skilled in the art.For example, " pact " can represent ± 10%, preferred ± 5%, more preferably ± 1%, most preferably ± 0.5%.
Description of drawings
Fig. 1 is C57/BL6 mice stress observed result figure;
Fig. 2 a is C57/BL6 mice walking platform gait analysis test result figure;
Fig. 2 b is C57/BL6 mice walking platform gait analysis test result figure;
Fig. 2 c is C57/BL6 mice walking platform gait analysis test result figure;
Fig. 2 d is C57/BL6 mice walking platform gait analysis test result figure;
Fig. 3 is spacious experiment test figure as a result.
The specific embodiment
For a better understanding and interpretation of the present invention, the present invention is described in further detail below with reference to accompanying drawings.
Embodiment 1
1, umbilical cord China's Tong Shi glue peels off
At once by the disconnected umbilicus intercepting of the conventional ligation of obstetrics umbilical cord, use the normal saline flushing umbilical cord after fetus is given birth to, the sterilization of reuse medical alcohol places umbilical cord to preserve 2-8 ℃ of constant temperature of liquid on umbilical cord and preserves.
Umbilical cord with the flushing of 0.9% sodium chloride injection obtains repeats 2～3 times, removes bloodstain.Whole umbilical cord sterilization of 75% ethanol submergence.The sodium chloride injection repeated washing is removed residual ethanol.Cut umbilical cord scissors into about 2～5cm number section with aseptic operation, remove congestion and grumeleuse in the umbilical cord segment blood vessel.Reject two tremulous pulsies of umbilical cord, a vein.White connective tissue between amniotic membrane and blood vessel is magnificent Tong Shi glue, with toothed forceps it is torn, and puts into aseptic plate, adds an amount of 0.9% sodium chloride injection, washing colloids.
2, piece of tissue preparation
Colloid is transferred to centrifuge tube, the weighing record.Cut with sterile tissue the magnificent Tong Shi colloid after weighing is cut into 1～4mm
3The tissue homogenate piece, add 0.9% sodium chloride injection, centrifugal 800～900g, 5min.It is aseptic to collect the censorship of last cleaning mixture.
3, inoculation
According to colloid weight, add an amount of culture medium, standardize solution tissue homogenate concentration is about 04～0.7g/ml, after pipettor piping and druming tissue homogenate piece is even, the tissue homogenate piece is inoculated in the T75 culture bottle, adds the culture medium Mixed culture.
4, the P0 of magnificent Tong Shi glue is commissioned to train foster
The horizontal culture bottle makes the tissue homogenate piece be uniformly distributed in whole bottom surface as far as possible, and culture bottle is positioned over carbon dioxide constant temperature and humidity incubator.Condition of culture: 37.0 ± 0.5 ℃, the carbon dioxide volume fraction is 5.0 ± 0.2%.
For the first time change liquid: piece of tissue is cultured to the 5th～7 day carries out full dose and changes liquid.Not adherent piece of tissue in the culture bottle and old culture medium merged together transfer to centrifuge tube, 800～900g, centrifugal 5min, remove supernatant, with the remnant tissue's piece after centrifugal, add the proper amount of fresh culture medium, pipet piping and druming is evenly, equivalent is all assigned to original culture bottle, adds culture medium then.The horizontal culture bottle makes piece of tissue be uniformly distributed in whole bottom surface as far as possible, places CO
2The constant temperature and humidity incubator continues to cultivate.
For the second time change liquid: cultivate and changed liquid on the 10th～13 day.The culture bottle that tilts slightly, pipet sop up the old culture medium of half amount gently, add the equivalent fresh culture.Place CO
2Incubator continues to cultivate.
Change liquid for the third time: when cultivating the 14th～16 day, if the cell fusion degree does not reach at 70%～80% o'clock, increase once by half amount and change liquid.Sop up the old culture medium of half amount gently with pipet, add the equivalent fresh culture again, place CO
2Incubator continues to cultivate.
5, cell harvesting
The 14th～18 day of cultivating of piece of tissue, the area percentage of cell clone group arrives at 70%～80% o'clock, the digestion results.Remove medium supernatant, 0.9% sodium chloride injection washed cell.Add an amount of digestive enzyme in the culture bottle, until soaking into the culture bottle bottom surface.After leaving standstill 1min, get culture bottle, observe under the inverted microscope, cell is rounded, and most of adherent piece of tissue and cell detachment stop digestion (digestion time is no more than 5min).Cell suspension moves in the centrifuge tube, a small amount of 0.9% sodium chloride injection rinsing bottle wall, and cleaning mixture changes in the centrifuge tube, after suspending, pipet piping and druming left standstill 30 seconds, the aseptic strainer filtering of 100 μ m, filtrate is centrifugal, 300g, 10min.Discard the washing supernatant, 0.9% sodium chloride injection re-suspended cell obtains P0 for cell.
Embodiment 2
1, P0 is for passage and cultivation
With cell suspension, centrifugal, 300g, 10min.Supernatant discarded is blown and beaten the resuspension cell with fresh culture, gently according to 5 * 10
3Individual/cm
2The density inoculation is gone down to posterity.Each T175 bottle graft kind cell suspension is added fresh culture.Being positioned over carbon dioxide constant temperature and humidity incubator begins to cultivate.Condition: 37.0 ± 0.5 ℃, the carbon dioxide volume fraction is 5.0 ± 0.2%.
2, passage
Cell culture to 72 ± 24 hour after the observation of cell degrees of fusion reaches 80%～90% under the inverted microscope mirror, can digest results, obtain P1 for cell.
2.1 digestion
It is standby to shift old culture fluid (as T175 culture bottle) to sterile chamber.An amount of 0.9% sodium chloride injection washes culture bottle gently, and cleaning mixture discards.Culture bottle adds for example trypsin digestion and cell of digestive enzyme in proportion, makes it to soak into the culture bottle bottom surface.After leaving standstill 1min, get culture bottle, observe under the inverted microscope, cell is rounded, and most of cell detachment stops digestion (digestion time is no more than 5min).The collecting cell suspension, an amount of 0.9% sodium chloride injection washing culture bottle, washing liquid is transferred in the centrifuge tube, and is centrifugal, 300g, 10min.
2.2 cell counting
Discard the washing supernatant, an amount of 0.9% sodium chloride injection re-suspended cell, resuspended liquid sample presentation detects cell counting and cell motility rate, and cell suspension is centrifugal, 300g, 10min.
2.3 go down to posterity and cultivate
Discard the washing supernatant, blow and beat re-suspended cell gently with fresh culture, with the packing behind the certain volume of re-suspended cell liquid standardize solution.Passage cell density inoculation is according to the rules gone down to posterity, and each T175 bottle graft kind cell suspension is added fresh culture.Place carbon dioxide constant temperature and humidity incubator to begin to cultivate.Condition: 37.0 ± 0.5 ℃, the carbon dioxide volume fraction is 5.0 ± 0.2%.
3, the results of cell and frozen
Cell culture to 72 ± 24 hour after the observation of cell degrees of fusion reaches 85%～90% under the inverted microscope mirror, can digest results, obtain P2 for cell.
3.1 digestion results:
With old culture fluid in the culture bottle be transferred in the sterile chamber (as the T175 culture bottle) standby.0.9% sodium chloride injection washed cell culture bottle discards cleaning mixture.Culture vessel adds trypsin digestion and cell, makes Digestive system soak into the culture bottle bottom surface.After leaving standstill 1min, get culture bottle and observe to inverted microscope, cell is rounded, and most of cell detachment stops digestion (digestion time is no more than 5min).The collecting cell suspension, 0.9% sodium chloride injection washing culture bottle, the cell suspension after the washing is transferred in the centrifuge tube, and is centrifugal, 300g, 10min.
3.2 washing:
Discard the washing supernatant, 0.9% sodium chloride injection re-suspended cell precipitation, the resuspended liquid of cell merges in the centrifuge tube, and is centrifugal, 300g, 10min.
3.3 filter and cell counting:
Discard the washing supernatant, 0.9% sodium chloride injection re-suspended cell precipitation.The resuspended liquid of cell is crossed 100 μ m cell screen clothes, elimination floccule or cell mass.Filtrate all moves in the centrifuge tube, and standardize solution, pipet are blown and beaten evenly gently, and sample presentation detects cell counting and cell motility rate, and filtrate equivalent branch installs in the centrifuge tube, add 0.9% sodium chloride injection standardize solution after, centrifugal, 300g, 10min.
3.4 the frozen packing of cell:
Abandoning supernatant according to count results, adds an amount of culture medium in centrifuge tube, re-suspended cell precipitates, and suspension volume standardize solution is arrived 1/2 of frozen final volume, and concentration is the twice of frozen final concentration, and the seed cell quality arbitration is carried out in sampling.Take out the cryopreserving liquid for preparing from 4 ℃ of refrigerators, slowly join in the cell suspension along tube wall, pipet is blown and beaten evenly gently, makes cell density and requires frozen consistent in density.Standard packing by every frozen pipe 1.0ml cell suspension.
3.5 procedural cooling:
The service routine cooling instrument carries out procedural cooling.
The cell that need are frozen is positioned in the procedural cooling instrument and is down to below-80 ℃ according to the frozen program of standard.
3.6 cell warehouse-in:
After the frozen end, freezing storing box taken out to transfer in the liquid nitrogen container store.
Embodiment 3
1, the recovery of cell
From liquid nitrogen container, take out frozen cell, put on the recovery frame of electric heating constant temperature tank (water temperature remains on about 40.0 ℃), rock rewarming fast.When becoming liquid state, namely finish by recovery by solid-state for cell suspension.
1.1 the washing of cell:
Cell suspension merges in the centrifuge tube.Add pre-cooling 0.9% sodium chloride injection, pipet piping and druming washing, standardize solution, mixing, centrifugal, 300g, 10min.
1.2 sampling counting and washing:
Discard the washing supernatant, an amount of pre-cooling 0.9% sodium chloride injection re-suspended cell precipitation is incorporated in the centrifuge tube, and sample presentation detects total cellular score and cell motility rate, and cell suspension is centrifugal, 300g, and 10min, supernatant discarded obtains P3 for cell.
1.3 go down to posterity and cultivate:
According to 5 * 10
3Individual/cm
2Passage cell density inoculation go down to posterity.Behind each T175 bottle graft kind cell suspension, add fresh culture.Being positioned over carbon dioxide constant temperature and humidity incubator begins to cultivate.Condition of culture: carbon dioxide constant temperature and humidity incubator, 37.0 ± 0.5 ℃, the carbon dioxide volume fraction is 5.0 ± 0.2%.
2, seed cell goes down to posterity
Cell culture to 72 ± 24 hour after the observation of cell degrees of fusion reaches 85%～90% under the inverted microscope mirror, can digest results, obtain P4 for cell.
2.1 digestion:
Remove medium supernatant, 0.9% sodium chloride injection washed cell, cleaning mixture discards.Add an amount of pancreatin in the culture bottle, until soaking into the culture bottle bottom surface.After leaving standstill 1min, get culture bottle, observe under the inverted microscope, cell is rounded, and most of attached cell comes off, and stops digestion (digestion time is no more than 5min).Cell suspension moves in the centrifuge tube, 0.9% sodium chloride injection rinsing bottle wall, and cleaning mixture is incorporated in the centrifuge tube, and is centrifugal, 300g, 10min.
2.2 cell counting:
Discard the washing supernatant, an amount of 0.9% sodium chloride injection re-suspended cell is incorporated into resuspended liquid in the centrifuge tube, and sample presentation detects cell counting and cell motility rate, and cell suspension is centrifugal, 300g, 10min.
2.3 go down to posterity and cultivate:
Discard the washing supernatant, blow and beat re-suspended cell gently with fresh culture, re-suspended cell liquid standardize solution is divided behind the certain volume be filled in the centrifuge tube.According to 5 * 10
3Individual/cm
2Passage cell density inoculation go down to posterity, behind each T175 bottle graft kind cell suspension, add fresh culture.Place carbon dioxide constant temperature and humidity incubator to begin to cultivate.Condition of culture: carbon dioxide constant temperature and humidity incubator, 37.0 ± 0.5 ℃, the carbon dioxide volume fraction is 5.0 ± 0.2%.
According to above-mentioned technology of the present invention, per 1 bottle of P0 is for cell (3 * 10
3), be passaged to P4 generation, can obtain 625 bottles, 3 * 10
6Cell/bottle.This is the excellent results that cell culture system of the present invention, culture process can be obtained.
The evaluation of embodiment 4 umbilical cord mesenchymal stem cells
1, cell surface antigen is identified
The P4 that embodiment 3 is prepared moves in the corresponding streaming pipe for the umbilical cord mesenchymal stem cells sample, adds the abundant mixing of 1ml PBS, and centrifugal 5 minutes of 500g abandons supernatant, repeated washing 2 times; Add an amount of PBS suspendible cell, filter, counting is adjusted into cell concentration (2.0～6.0) * 10
6Individual/ml is stand-by; Take out some streaming pipes, add and respectively organize antibody and homotype contrast agents (lucifuge operation); Then ready cell sample adding has been added in the streaming pipe of antibody reagent, every pipe 100 μ l, fully mixing is put 4 ℃ of refrigerator lucifuges and was hatched 30 minutes; Hatch that every pipe adds 1ml PBS after finishing, abundant mixing, centrifugal 5 minutes of 500g abandons supernatant, repeats this washing process 1 time, adjusts about cell suspension volume to 200 μ l last machine testing.
The result is as shown in the following table 1.
| Label | CD105 | CD90 | CD73 | CD44 | CD29 | CD45 | CD34 | CD19 | HLA-DR | CD14 |
| Content | 99.8% | 99.9% | 99.2% | 99.3% | 99.9% | 1.4% | 0.7% | 1.0% | 0.7% | 1.0% |
The result shows: the CD90 of the umbilical cord mesenchymal stem cells of acquisition, CD29, CD73, CD105 express all greater than 99%, and simultaneously, CD45, CD34, CD19, CD14, HLA-DR express all less than 2%.
2, skeletonization becomes fat to induce differentiation potential to identify
2.1 becoming the fat differentiation identifies
The P4 that embodiment 3 is prepared is l * 10 for cell sample adjustment cell density
5Ml is inoculated in 24 orifice plates, and cell fusion reaches 70%～80% after 1～2 day, and cultivate with lipoblast inducing culture liquid this moment, and full dose was changed liquid 1 time in per 3 days, oil red 0 dyeing in the 21st day.
Cell sample becomes fat to induce behind the 21st day oil red O stain fat to drip to take on a red color, and matched group fat then do not occur and drips.About 30-40% is converted into adipose cell in the umbilical cord mesenchymal stem cells.
2.2 Osteoblast Differentiation is identified
The P4 that embodiment 3 is prepared is l * 10 for cell sample adjustment cell density
5/ ml is inoculated in 24 orifice plates, and cell fusion reaches 70%～80% behind 1～2d, and cultivate with osteoblast inducing culture liquid this moment, and full dose was changed liquid 1 time in per 3 days, alizarin red dyeing in the 28th day.
The result shows: osteogenic induction alizarin red dyeing in the 28th day back calcification tuberosity takes on a red color, and matched group does not then occur.Through observing, surpass 70% in the umbilical cord mesenchymal stem cells and be converted into osteoblast.
Embodiment 5 Research of Animal Model for Study
With 1-methyl-4-phenyl-1,2,4, the mice that 6-tetrahydropyridine (MPTP) brings out the parkinson disease symptom be scale-model investigation human umbilical cord mesenchymal stem cells of the present invention to treating Parkinsonian effectiveness and effective dosage ranges.
1, laboratory animal
The C57/BL6 mice, 25-28g, two monthly ages, free diet.Get 45 of mices, be divided into 3 groups at random, i.e. blank group (giving normal saline), (carry out MPTP as described below induces the PD model group, tail vein injection saline) and stem-cell therapy group (carry out MPTP as described below and induce, then the tail vein injection stem cell).
2,1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine (MPTP) is induced
C57/BL6 mouse peritoneal injection MPTP, 40mg/Kg injected 5 days continuously.
3, stem-cell therapy
The last time MPTP injection back the 6th day to the C57/BL6 mice by tail vein injection, by 1 * 10
6Cell/kg dosage gives P4 that embodiment 4 obtains for umbilical cord mesenchymal stem cells.
The PD model group is the 6th day injecting normal saline in MPTP injection back the last time.
4, behavioristics is detected
(1) walking platform gait analysis test
Making animal PD model (being that MPTP induces) preceding 7 days, mice is being carried out walking platform gait analysis (Catwalk) test training.Allow and respectively organize the passage of mice exercise by substituting, walk back and forth every day 6 times, trained continuously 7 days.After training finished, each organizes mice all can not pass through all disallowable experiments of mice of passage continuously continuously without interruption by passage.Utilize commercially available walking platform (Catwalk) animal gait analysis system platform to observe and the characteristic index of quantitative analysis mice by the walking platform, wherein allow and respectively organize mice by the CatWalk passage.Described animal gait analysis system platform comprises glass floor and sealing up and down, two ends have the walking platform of the passage of entrance and exit, the glass light source, photographic head, image pick-up card and analytical equipment, when mice is passed by the walking platform along passage, the glass floor that the claw contact is illuminated by the glass light source, the pawl seal that stays is sent into image pick-up card after being photographed by the photographic head of walking platform below, analyze these pawl seals and can obtain going on foot order correlation time by analytical equipment, gait, duration, the step-length of each claw, distance between the every pair of fore paw and the rear solid end, the length width of pawl seal, the pressure and other parameters of claw, and add up or calculate two metapedes spacings and paces cycle significant prolongation thus, wave indexs such as speed and stride.By to described parameter and statistics or calculating, can carry out quantitative analysis to described index.
In this test, each organizes mice MPTP injection back the 21st day the last time, observes and the characteristic index of quantitative analysis mice by the walking platform, wherein allows respectively organize mice and pass through the CatWalk passage every mice detection 3-4 time.Parameter and the index of test comprise:
Two metapedes pawl spacings (Base of support): two metapedes are simultaneously during the support glass floor, distance between the two.
The paces cycle (Step cycle): in the walking process, during same foot contact glass floor, the cycle at interval.
Wave speed (Swing speed)=stride/rolling period.
Stride (Stride length): the distance in the walking process between the double kiss the earth of the heel/toe of same foot.
Rolling period: each paces is in the cycle, the time that the animal foot is unsettled.
(2) spacious experiment test (Open field test)
Spacious experiment is a kind of method that detects the animal movement function on the whole, detection be the total distance of animal walking at the appointed time.
In this experiment, last MPTP injection back the 7th day, each is organized mice and does spacious experiment.Each is organized mice head places 50cm * 50cm towards same corner the spacious field of polyethylene, observed continuously 10 minutes.Detect the distance respectively organize walking in the mice 10 minutes.
5, test result
(1) stress of mice
The behavior of respectively organizing mice is observed in last MPTP injection back the 7th day.
The phenomenon that accusing each other appears in PD model group mice.In the stem-cell therapy group mice, behind stem-cell therapy, the degree that mice accuses each other reduces.As shown in Figure 1, serious wound appears in PD group mice whole body, and behind the stem-cell therapy, the wound situation of mice alleviates to some extent.
(2) gait of mice
Shown in Fig. 2 a-2d, after the MPTP injection, the two metapedes spacings of mice and paces cycle significant prolongation wave speed and stride obviously reduces.Behind the stem-cell therapy, the two metapedes spacings of mice and paces cycle significantly reduce, and wave speed and stride significantly raises.
(3) voluntary activity of mice
After the MPTP injection, the distance that mice walked in 10 minutes significantly reduces.Behind the stem-cell therapy, the distance of mice walking significantly increases.
The present inventor finds can treat for example parkinson disease of neurodegenerative diseases effectively with the umbilical cord mesenchymal stem cells of method preparation of the present invention unexpectedly.The umbilical cord mesenchymal stem cells that utilizes the present invention to prepare has the advantage that is different from prior art: a little less than (1) immunogenicity, and induction of immunity rejection not; (2) have the characteristic of similar " immunity exempt ", can suppress the alloimmunity reaction, the anti-host disease of inhibition of transplant (GVHD); (3) do not relate to society, ethics, legal more arguements; (4) source is sufficient, and patient's body constitution is not had very high request, and general patient is acceptant; (5) separation method is simple, and cell viability is very strong, and easily a large amount of amplifications may to have a longer telomere relevant with it; (6) umbilical cord mesenchymal stem cells is a kind of more original population of stem cells.Umbilical cord mesenchymal stem cells provided by the invention can be treated parkinson disease effectively.
Unless otherwise noted, practice of the present invention will be used the routine techniques of biotechnology, organic chemistry, inorganic chemistry etc., obviously except the special description of institute in above-mentioned explanation and embodiment, can also other mode realize the present invention.Other aspect within the scope of the present invention will be apparent to those skilled in the art in the invention with improvement.According to instruction of the present invention, many changes and variation are feasible, so it within the scope of the present invention.All patents mentioned in this article, patent application and technical paper all are attached to this paper accordingly by reference.
Claims (10)
1. the application of umbilical cord mesenchymal stem cells in the Parkinsonian preparation of preparation treatment, described umbilical cord mesenchymal stem cells prepares by the following method:
Step 1: obtain umbilical cord China Tong Shi glue;
Step 2: piece of tissue preparation and inoculation, wherein described colloid is sheared into about 1～4mm
3The tissue homogenate piece, be about 0.4～0.7g/ml with serum-free medium standardize solution tissue homogenate concentration, be inoculated in the culture bottle after the piping and druming homogenate evenly, add serum-free medium and cultivate;
Step 3:P0 is for cell culture, wherein above-mentioned culture bottle is positioned over CO2 gas incubator and cultivates, and is cultured to the 5th～7 day and carries out full dose to change liquid; Be cultured to the 10th～13 day and carry out half amount and change liquid; Be cultured to the 14th～18 day, the cell fusion degree reaches at 70%～80% o'clock, obtains P0 for cell with digestive enzyme digestion results;
Step 4:P1 is for cell culture, wherein with described P0 for cell according to about 5 * 10
3Individual/cm
2Density is inoculated in the culture bottle, is positioned over CO2 gas incubator and cultivates, and cell culture to the 2～4 days after the cell fusion degree reaches 80%～90%, obtains P1 for cell with digestive enzyme digestion results;
Step 5:P2 is for cell culture, wherein with described P1 for cell according to about 5 * 10
3Individual/cm
2Density is inoculated in the culture bottle, and be positioned over CO2 gas incubator and cultivate, cell culture to the 2～4 days, the cell fusion degree reaches 85%～90%, obtains P2 for cell with digestive enzyme digestion results;
Step 6:P3 is for cell culture, wherein with described P2 for cell inoculation in culture bottle, be positioned over CO2 gas incubator and be cultured to the cell fusion degree and reach〉after 95%, obtain P3 for cell with digestive enzyme digestion results;
Step 7:P4 is for cell culture, wherein with described P3 for cell according to about 5 * 10
3Individual/cm
2Density is inoculated in the culture bottle, is positioned over CO2 gas incubator and cultivates, and cell culture to the 2～4 days after the cell fusion degree reaches 85%～90%, obtains the P4 umbilical cord mesenchymal stem cells in generation with digestive enzyme digestion results.
2. application according to claim 1, wherein step 5 also comprises the described P2 that digestive enzyme digestion results are obtained for cell cryopreservation, wherein with described P2 for cell suspension in cryopreserving liquid, be positioned in the procedural cooling instrument be down to below-80 ℃ frozen.
3. application according to claim 2, wherein step 5 also comprises frozen P2 for cell recovery, wherein with described frozen P2 for cell in the about 40.0 ℃ of following water-baths of temperature, rock rewarming fast, cell suspension, is seeded in the Tissue Culture Flask when becoming liquid state by solid-state.
4. application according to claim 1, wherein cell culture to the if the cell fusion degree does not reach 70%～80%, carried out once half amount again and changes liquid in the time of 14～16 days in the step 3.
5. application according to claim 1, wherein said digestive enzyme are trypsin.
6. according to each described application among the claim 1-5, wherein in the cell culture of step 3-7, all adopt serum-free medium to carry out cell culture.
7. according to each described application among the claim 1-6, wherein also comprise step 8, wherein the umbilical cord mesenchymal stem cells to described P4 generation carries out the surface antigen check, screening surface antigen CD90, CD29, CD73, CD105 express greater than 99%, and surface antigen CD45, CD34, CD19, CD14, HLA-DR express the umbilical cord mesenchymal stem cells less than 2%, and/or
Umbilical cord mesenchymal stem cells to described P4 generation carries out the skeletonization conversion ratio and becomes the fat conversion ratio to detect, and screening skeletonization conversion ratio is greater than 70%, and becomes the fat conversion ratio to be the umbilical cord mesenchymal stem cells of about 20-30%.
8. one kind is used for the treatment of Parkinsonian preparation compositions, wherein contains umbilical cord mesenchymal stem cells, described umbilical cord mesenchymal stem cells in the claim 1-7 definition.
9. preparation compositions according to Claim 8, the dosage of wherein said umbilical cord mesenchymal stem cells is about 1 * 10
6Cell/kg～9 * 10
6Cell/kg, preferred, the amount of described umbilical cord mesenchymal stem cells is about 1 * 10
6Cell～9 * 10
6Cell.
10. according to Claim 8 or 9 preparation compositions, surface antigen CD90, the CD29 of wherein said umbilical cord mesenchymal stem cells, CD73, CD105 express greater than 99%, and surface antigen CD45, CD34, CD19, CD14, HLA-DR express less than 2%,
And/or,
The skeletonization conversion ratio of wherein said umbilical cord mesenchymal stem cells is greater than 70%, and one-tenth fat conversion ratio is about 20-30%.
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| WO2018149127A1 (en) * | 2017-02-16 | 2018-08-23 | 上海安集协康生物技术股份有限公司 | Neural stem cell agent for treatment of parkinson's disease by intranasal administration, preparation method and application thereof |
| CN109468273A (en) * | 2018-12-29 | 2019-03-15 | 厦门博瑞思生物科技有限公司 | A method of obtaining mescenchymal stem cell from umbilical cord |
| CN110438071A (en) * | 2019-08-26 | 2019-11-12 | 广东唯泰生物科技有限公司 | People's umbilical cord China Tong Shi glue mesenchymal stem cells and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018149127A1 (en) * | 2017-02-16 | 2018-08-23 | 上海安集协康生物技术股份有限公司 | Neural stem cell agent for treatment of parkinson's disease by intranasal administration, preparation method and application thereof |
| CN109468273A (en) * | 2018-12-29 | 2019-03-15 | 厦门博瑞思生物科技有限公司 | A method of obtaining mescenchymal stem cell from umbilical cord |
| CN110438071A (en) * | 2019-08-26 | 2019-11-12 | 广东唯泰生物科技有限公司 | People's umbilical cord China Tong Shi glue mesenchymal stem cells and preparation method thereof |
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