CN103849595A - Large scale production technology for dental pulp stem cell - Google Patents
Large scale production technology for dental pulp stem cell Download PDFInfo
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- CN103849595A CN103849595A CN201210514497.XA CN201210514497A CN103849595A CN 103849595 A CN103849595 A CN 103849595A CN 201210514497 A CN201210514497 A CN 201210514497A CN 103849595 A CN103849595 A CN 103849595A
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Abstract
The invention provides a large scale production technology for a dental pulp stem cell. The large scale production technology comprises following steps of (1) separating and extracting the dental pulp stem cell; (2) amplifying the dental pulp stem cell through two dimension cultivation; (3) detecting the dental pulp stem cell; (4) cultivating the dental pulp stem cell on a large scale; (5) freeze preserving the dental pulp stem cell; and (6) freeze preserving the dental pulp stem cell for a long term. The invention provides an in vitro large-scale cultivating and amplifying method for a dental pulp stem cell, establishes a long-term, standard and systematized freeze preserving system for a high quality high activity dental pulp stem cell, so that the method can provide a high enough quality stem cell resource for clinic and scientific researches, and has low cost and a wide application prospect.
Description
Technical field
The invention belongs to biomedical engineering field, be specifically related to cultivation amplification and the freezing and storing method of stem cell, more specifically relate to the scale production process of dental pulp stem cell.
Background technology
1999, " Science " is chosen as hESC's achievement in research then first of the large Progress & New Products in the world ten, 2000, " Time " weekly is classified as first of 20 end of the century world's ten large technological achievements, and thinks that embryonic stem cell and human genome will become the field of tool development of new millennium and application prospect simultaneously.So far stem-cell research becomes in recent years in biology and medical field one of the most noticeable focus.Stem cell is the initiating cell that plays " trunk " effect in the g and D of biont, is a kind of cell colony with self, highly propagation and multi-lineage potential, and it comprises embryonic stem cell and adult stem cell.Although embryonic stem cell can be divided into various cell types, this differentiation is " non-polarization ", and has the problem of the aspects such as ethics.Adult stem cell does not exist the problems such as ethics, law, immunological rejection, there is the advantage such as wide of drawing materials conveniently, originate simultaneously, thereby adult stem cell has good potential applicability in clinical practice in the research of organizational project, gene therapy and individualized treatment.
The defect of teeth and the absence of tooth that cause due to a variety of causes have become the ubiquitous disease of one that endangers oral cavity and even HUMAN HEALTH, and its sickness rate is the trend increasing year by year.Mainly fill and deal with tooth defect disease by means of various dental materials at present, although can stop to a certain extent the continuation development of pathology, can not solve root problem.For absence of tooth, although solution route is a lot, the most effective and the most basic way or the biological regeneration of tooth.
Along with the fast development of tissue regeneration medical science, for effective treatment of defect of teeth and absence of tooth has brought hope.Dental tissue has been born at present existing many research and utilization Dental epitheliums and the mesochymal interaction of property of tooth source again.Conventional tooth source property mesenchyme comprises dental papilla cells and Dental Pulp Cells.Because dental papilla cells is difficult to obtain clinically, therefore at present for the cell in the first-selected pulp tissue of the seed cell source of tooth body and regeneration of tooth, wherein dental pulp stem cell most importantly.
Within 2000, find first dental pulp stem cell (S.Gronthos; M.Mankani; J.Brahim; P.Gehron Robey; and S.Shi; Postnatal human dental pulp stem cells (DPSCs) invitro and in vivo, Proc Natl Acad Sci USA.2000 December 5; 97 (25): 13625-13630.).Dental pulp stem cell is the cell that a class has the stem cell characteristics such as high proliferation ability, height self-renewal capacity, Multidirectional Differentiation ability, is therefore the desirable seed cell that builds tooth body and dental tissue.Existing this cell construction dental pulp of research and utilization, dentine and dental tissue.
The discovery of dental pulp stem cell, for engineering of biological tooth has been brought breakthrough progress.But the engineering science with other tissue is the same, as quick obtaining and the rapid amplifying of the dental pulp stem cell of seed cell, be a bottleneck of engineering of biological tooth development.
In dental pulp stem cell process of clinical application, distinct issues are once successfully to treat the cell that needs some amount at present, this means that obtaining how at short notice the highly active cell of mass efficient is a very crucial problem to offer patient's use.Therefore, the research of external a large amount of amplification dental pulp stem cells is more and more subject to researchist and clinician's attention.There is at present the method and apparatus of some expanding stem cells, but traditional two dimensional surface expanding stem cells method, more difficultly obtain at short notice enough cell concentrations, and passage number repeatedly can cause stem cell quality to reduce, with respect to powerful clinical demand, its quantity is also nowhere near.
And on the other hand, from deciduous teeth to permanent teeth, replace, impacted tooth, accessory tooth pull out and abnormal tooth proofread and correct, a large amount of teeth go out of use, dental pulp stem cell resource wherein runs off in vain.Therefore, invent a kind of method that dental pulp stem cell long-term frozen preserves significant equally.
The cultivation of traditional anchorage-dependent cell adopts the cultural methods such as static or rolling bottle, and complicated operation, expends space and manpower.1967, Van Wezel has proposed the concept of " micro-carrier system " first, for the high-density culture of anchorage-dependent cell provides thinking, and create bio-reactor microcarrier culturing cell technique, making animal cell culture enter high-density culture stage microcarrier refers to by sephadex or other polymkeric substance and forms microballon, diameter generally, between 60-200 μ m, is applicable to the growth of anchorage-dependent cell.Use this type of bead to provide area of attachment for cell, by stirring carrier is suspended in nutrient solution gently, cell also becomes suspended state, and this training method is called as Microcarrier Culture Techniques.The microcarrier using at present has business-like Cytodex 1/3, Cultispher G/S, Biosilon, Hillex and HyQSp, and homemade Mierocrystalline cellulose, pottery, chitosan, collagen, gelatin, PCL, PLA, PLGA etc.Due to the shared volume of microcarrier itself and quality little, but have very large useful area can supply cell attachment, thereby greatly improved production efficiency.Such as 5mg Cytodex 1 surface-area can reach 30cm
2/ mL, and traditional monolayer culture is difficult to reach so high surface-area/volumetric ratio.1L microcarrier is cultivated the cell of producing and is equivalent to the cell that 50 rolling bottles are produced.This training method also declined many to labor force's demand, save cleaning and the preparation work of Glass Containers etc.; Cell is simple with separating of nutrient solution, stops once stir, and after 3min, cell can rely on its gravity and sedimentation, without carrying out centrifugally operated, has reduced complicated operation steps.And cellular physiological activity culture parameters etc. are easily controlled, contamination probability also reduces greatly.
Major obstacle in extensive stem cell culturing process comprises restriction, byproducts build-up, more intelligent process control, the problems such as the extreme sensitivity of zooblast to mechanical shear stress of supporting.
Stem cell clinical treatment needs a large amount of highly active stem cells; so the focus of how to cultivate in a short time in vitro the high-quality treatment cell of sufficient amount and providing a mass-producing, stdn, systematized cell stocking system to become academia and industrial community close attention is also at the early-stage about this research on the one hand at present.
Summary of the invention
The present invention is directed to quick obtaining, amplification and the long-term problems such as difficulty of preserving of the existing dental pulp stem cell as seed cell, find one and cultivate more efficiently amplification and long-term preservation method.The present invention uses bio-reactor microcarrier to cultivate a large amount of expanding stem cells of dental pulp stem cell technology, and the stdn of having set up dental pulp stem cell, the frozen system of systematize, solve this class resource and can not get the present situation reusing, and set up cell bank that long-term high quality stores dental pulp stem cell and provide enough high-quality stem cell resources to facilitate as clinical and scientific research, with low cost, have a extensive future.
The scale production process of dental pulp stem cell provided by the invention comprises the steps:
(1) separation and Extraction of dental pulp stem cell: by pulp tissue with after collagenase digesting, containing carrying out in the culture vessel of foetal calf serum cell culture fluid, in 37 DEG C, 5%CO
2under condition, cultivate, within 3 days, change liquid;
(2) cultivation of dental pulp stem cell amplification: the primary cell that step (1) is cultivated to gained is with 0.1 × 10
5-1.0 × 10
5individual cell/cm
2density be inoculated in two-dimentional culture vessel, add fresh cell culture fluid, in 37 DEG C, 5%CO
2under condition, cultivate, within 3 days, change liquid;
(3) detection of dental pulp stem cell: carry out cytoactive detection, sterility detection and fluidic cell and detect;
(4) large scale culturing of dental pulp stem cell: adopt spinner culture or bioreactor culture, after dental pulp stem cell is seeded in rolling bottle or bio-reactor, adjust dissolved oxygen concentration, pH value, mixing speed and air flow, change substratum according to the result of sampling analysis cell counting, survival rate, nutritive ingredient and metabolite, thereby reach the high-density large-scale cultivation of cell;
(5) the freezing preservation of dental pulp stem cell: will detect qualified cell cryopreservation in pipe, the details of cell are scanned into data management system, automation system is according to liquid nitrogen situation Automatic-searching vacant locations, by cell cryopreservation in corresponding space;
(6) as required, carry out the long-term frozen of dental pulp stem cell and preserve, between preservation period, regularly from frozen environment, take out the cell detection of recovering.
In step (1), pulp tissue selects under aseptic condition, after rinsing, shreds with damping fluid; The digestion of pulp tissue adopts 0.1% ~ 0.6% collagenase of 5 ~ 10 times of volumes, in 37 DEG C of incubators, after shaking culture 30 ~ 180min, stop digestion with the cell culture fluid containing foetal calf serum, described cell culture fluid commercial α-MEM, DMEM substratum and serum free medium containing 10-20% foetal calf serum.In these cell culture fluids, contain support stem cell growth sugar, amino acid, somatomedin, inorganic salt, trace element etc.
Culturing step in step (1) comprises and will after the centrifugal 3 ~ 10min of 800 ~ 1500r/min, abandon supernatant, add 5 ~ 10 times of volume cell culture fluid re-suspended cells through the pulp tissue of digestion, proceed in culture vessel and cultivate, described cell culture fluid commercial α-MEM, DMEM substratum and serum free medium containing 10-20% foetal calf serum.In these cell culture fluids, contain support stem cell growth sugar, amino acid, somatomedin, inorganic salt, trace element etc.
In step (2), primary cell digests and blows and beats cell with pancreatin solution in the time covering with culture vessel bottom 80%-90%, is inoculated in two-dimentional culture vessel after making cell depart from primary culture vessel completely.Described two-dimentional culture vessel can be selected from cell cultures orifice plate, culture dish and tissue culture square vase.
Spinner culture described in step (4) is undertaken by following process: by microcarrier after pre-treatment, rolling bottle silication, 121 DEG C of high-temperature sterilizations, inoculating cell, microcarrier add-on is 2-10g/L, magnitude range is 60 ~ 300 μ m, inoculum density is 3 × 10
4-3 × 10
5individual cell/ml, the magnitude range of rolling bottle is 25ml-500ml, speed range 50-75rpm.
Bio-reactor described in step (4) is selected from biological reactor for cell culture, comprises stirring type bioreactor, fixed-bed bioreactor, WAVE bio-reactor etc.
In production technique of the present invention, the stirring type bioreactor, the WAVE bio-reactor that use carry out cell cultures by following process: microcarrier PBS is rinsed 2 times, adding for the third time PBS to put into 4 DEG C of refrigerator inner equilibrium aquations spends the night, after high pressure steam sterilization, inoculation dental pulp stem cell, the add-on of microcarrier is 3-10g/L, and inoculum density is 1 × 10
5-10
6individual cell/mL, cell amplification to density is 5 × 10
5-2 × 10
7individual cell/mL.In production technique of the present invention, the fixed-bed bioreactor using carries out cell cultures by following process: in bio-reactor, add polyester slice through surface treatment well cutting as cell carrier, and add phosphoric acid buffer, after high-temp steam sterilizing, add cell culture fluid, inoculation dental pulp stem cell, wherein the usage quantity of trevira carrier is every liter of tank volume 25-45g, and inoculum density is 1 × 10
5-10
6individual cell/mL, cell amplification to density is 5 × 10
5-2 × 10
7individual cell/mL.Described in above-mentioned bioreactor culture condition, dissolved oxygen concentration is that 40%-60%, pH value are that 7.0-7.2, mixing speed are 50-100rpm.
In production technique of the present invention, in step (5), the cell details of input comprise donor name, cell algebraically, cell culture fluid composition and concentration, cell inoculum density, frozen storing liquid composition and frozen date; Described freezing preservation comprises obtained dental pulp stem cell by 1 × 10
6~ 1 × 10
7individual/ml cell density is resuspended in frozen storing liquid, is distributed into cryopreservation tube, after programmed cooling, cryopreservation tube is transferred to freezing preservation in-196 DEG C of liquid nitrogen.Described frozen storing liquid comprises DMSO, foetal calf serum and α-MEM nutrient solution 1:2:7(volume ratio).
As required, carry out the long-term frozen of dental pulp stem cell and preserve, between preservation period, regularly from frozen environment, take out the cell detection of recovering.
Detailed Description Of The Invention
Describe dental pulp stem cell scale production process of the present invention below in detail.
One, the separation and Extraction of dental pulp stem cell
Fresh donor dental tissue is adopted containing antibiotic buffer salt solution and completes transfer, under aseptic condition, select pulp tissue and rinse with damping fluid; The pulp tissue picking out is shredded, add 0.1% ~ 0.6% collagenase of 5 ~ 10 times as Digestive system, dispel evenly with suction pipe, be placed in 37 DEG C of incubator shaking culture 30 ~ 180min.After having digested, in suspension, add containing α-MEM nutrient solution of 15% foetal calf serum and stop digestion, at the centrifugal 5 ~ 10min of 800 ~ 1500r/min, abandon after supernatant, add 5 ~ 10 times of volumes containing α-MEM nutrient solution re-suspended cell of 15% foetal calf serum, proceed to and in culture vessel, carry out former culture.
Two, the cultivation of dental pulp stem cell amplification
At the bottom of primary cell covers with vessel 80 ~ 90%, digest and blow and beat cell with the pancreatin solution containing 0.25%EDTA, after cell is departed from completely, adopt different training method renewed vaccinations, and add cell culture fluid, in constant incubator, cultivate and increase.
Cultivate when amplification when adopting two-dimentional culture vessel to carry out dental pulp stem cell, according to the quantity of the bottom surface area of culture vessel and primary cell, by primary cell with 0.1 × 10
5-1.0 × 10
5individual cell/cm
2density be inoculated in two-dimentional culture vessel, add cell culture fluid α-MEM nutrient solution of 15% foetal calf serum (for example containing) in 37 DEG C, 5%CO
2under condition, cultivate, within 3 days, change liquid.
Two dimension culture vessel common are various cell cultures orifice plates, Petri culture dish, tissue culture square vase etc.
Three, the detection of dental pulp stem cell
After dental pulp stem cell extracts, carry out cytoactive detection, sterility detection, fluidic cell detection etc.
Cytoactive detects, and comprises observation of cell growing state under the microscope, and cell is inoculated in 24 orifice plates, makes cell growth curve by cck-8 legal system.
Sterility detects, and collects the nutrient solution of cultivating cell, after cultivating, examines under a microscope and carries out fungi, Bacteria Detection in the biochemical cultivation case of 25 DEG C, 37 DEG C.
Fluidic cell detects, and detects the specificity of cell, comprises mescenchymal stem cell specific surfaces mark Stro-1, CD29, CD44, CD71, CD90, CD106, CD120 etc., and other is cell surface marker, as CD14, CD34, CD45 etc.
Four, the large scale culturing of dental pulp stem cell
Adopt spinner culture or bio-reactor to carry out the large scale culturing of dental pulp stem cell, in culturing process, should adjust dissolved oxygen concentration, pH value, mixing speed and air flow, change substratum according to the result of sampling analysis cell counting, survival rate, nutritive ingredient and metabolite, thereby reach the high-density large-scale cultivation of cell.
A subject matter of bioreactor culture is exactly dissolved oxygen restriction, the solubleness of the oxygen in substratum very low (7.6 μ g/ml), typical density 2 × 10
6/ ml cultured cells will exhaust the oxygen in substratum within the time less than 1h, therefore must in culture cycle, constantly in substratum, pass into oxygen.The dissolved oxygen maintaining in nutrient solution by the ratio of control air, nitrogen, carbonic acid gas, oxygen supplies cell consumption a suitable level.In culturing process, make cell suspension in substratum by stirring on the one hand, can promote on the other hand liquid mixing mass transfer, improve oxygen delivery capacity.Thereby the byproducts build-up of cellular metabolism can affect the pH value of nutrient solution has influence on Growth of Cells, thus need to add by analyzing medium component control the speed of fresh culture, to offer good nutrient environment of cell.
The process of spinner culture amplification comprises: by microcarrier after pre-treatment, rolling bottle silication, 121 DEG C of high-temperature sterilizations, inoculating cell, microcarrier add-on is 2-20g/L, inoculum density is 3 × 10
4-3 × 10
5individual cell/ml, the magnitude range of rolling bottle is 25ml-500ml, the magnitude range of microcarrier is 60 ~ 300 μ m, speed range 50-75rpm/min, training method is batch culture.
The process of bioreactor culture amplification comprises: the 1. pre-treatment of microcarrier: microcarrier PBS is rinsed 2 times, add for the third time PBS to put into 4 DEG C of refrigerator inner equilibrium aquations and spend the night, high pressure steam sterilization; 2. the dental pulp stem cell of rolling bottle or a large amount of two-dimentional plate amplification cultivation is made to cell suspension with the α-MEM nutrient solution containing 15% foetal calf serum, be seeded on microcarrier, the volume of reactor is 3-10L, and the add-on of microcarrier is 3-10g/L, and inoculum density is 1 × 10
5-10
6individual cell/mL, it is 5 × 10 that cell cultures is expanded to density
5-2 × 10
7individual cell/mL; 3. gather in the crops with the pancreatin containing EDTA the dental pulp stem cell increasing, carry out subsequent detection.
Bioreactor culture amplification can be one-level bioreactor culture, can be also multistage cultivation.In one-level bio-reactor, Growth of Cells is to a certain extent time, according to carrying out next stage cell amplification culture to the requirement of cell quantity, by after the cell complete digestion on microcarrier as seed cell inoculation next stage bio-reactor or directly add new microcarrier and carry out ball and turn ball amplification culture.
Bio-reactor can be selected from stirring type bioreactor, fixed-bed bioreactor, wave (wave) bio-reactor etc., and other various forms and cells in vitro large scale culturing system or device based on various principles.
Stirring type bioreactor is mainly to rotate to stir nutrient solution to increase mass transfer ability by agitator, guarantee the homogeneity of oxygen concn and the nutritive substance of cell cultures, reach the object of large scale culturing cell, make nutrient solution apply certain shearing stress to cell simultaneously.At present the most frequently used in medical tissue engineering research is blender jar reactor.Its principal feature is to adopt magnetic force rotor to stir nutrient solution, stem cell is seeded in the nutrient solution of magnetic agitation, and cell is grown in the nutrient solution of prolonged agitation.The great advantage of stirring type bioreactor is, can cultivate various types of zooblasts, and culture process easily amplifies, and constant product quality is very suitable for batch production and produces.
The inner chip carrier of filling of fixed-bed bioreactor, makes the growth of cell docile, makes cell reach higher density by continous pouring fresh medium, in culturing process, cytotrophy abundance, metabolic waste can be removed in time, for Growth of Cells provides a good environment.The chip carrier using comprise NBS company trevira etc. all can be beneficial to the material of cell docile growth.
WAVE bio-reactor is disposable wave bag bio-reactor, in order to substitute traditional stainless steel fermentor tank.One-time reaction device is applicable to various types of cell cultures.Gentle undulation can play the object of good oxygen supply and mixing, and meanwhile, the shearing force that this mode of motion produces is also very little, is far smaller than in traditional tank body by the shearing force stirring or air lift type method produces; Can support high-density large-scale cell cultures, and can not form the destruction of foam and shearing force.
The pattern that adopts above bio-reactor to carry out cell cultures comprises batch culture pattern, intermittent type training mode and continous pouring formula training mode.
Batch culture pattern is that crushing stem cell and disposable packing in bio-reactor of nutrient solution are cultivated, and cell is constantly grown, amplification in a large number, after after a while, this reactive system is taken out, and digestion obtains high-quality dental pulp stem cell.
Intermittent type training mode adopts intermittent type bio-reactor, in reactor, add nutrient solution, carry out after sterilizing, access cell strain carries out cell cultures under the condition that maintains applicable Growth of Cells and product generation, in reaction process except needs pass into a certain amount of sterile air, defoamer and soda acid, generally no longer add substratum and culture, also do not take out product, after the reaction such as only having to proceed to a certain degree, just all nutrient solution is emitted, carry out aftertreatment.
Continous pouring formula training mode is installed cell microcarrier device for trapping in continous pouring formula bioreactor system, and cell seed is added in reactor and cultivated together with nutrient solution.Fresh medium constantly adds in reactor on the one hand; Again reaction solution is continuously taken out on the other hand, make reaction conditions in a kind of steady state.Continous pouring formula bio-reactor can carry out more accurate monitoring and control the microenvironment of cell, both time saving and energy saving, has reduced again cell the chance of polluting occurs, and the density of culturing cell and quality can be greatly improved.
Five, the freezing preservation of dental pulp stem cell
After cell harvesting, repeatedly blow and beat cell with nutrient solution, mix, send into medicine non-clinical study quality control procedure standard laboratory and carry out quality examination, comprise aseptic detection, detection of mycoplasma, virus detects, and immunity table shape is detected, cell technology and vitality test, biological efficacy detects, and cytodifferentiation ability detects to determine whether it arrives criterion of acceptability.Through detecting standard compliant dental pulp stem cell by 1 × 10
6~ 1 × 10
7the density of individual cell/ml is resuspended in frozen storing liquid DMSO, foetal calf serum and α-MEM nutrient solution 1:2:7, is distributed into cryopreservation tube.
Fill in the details of cell, comprise donor name, cell algebraically, cell culture fluid composition and concentration, cell inoculum density, frozen storing liquid composition and frozen date etc., the true freezing situation of each sample of complete documentation, information induction position alignment scanning instrument on cryopreservation tube, information is scanned into data management system like clockwork, completes cell bank Data Enter and structure.
Each cryopreservation tube, after programmed cooling, is transferred to freezing preservation in-196 DEG C of liquid nitrogen.System is according to liquid nitrogen situation Automatic-searching vacant locations, by cell cryopreservation in corresponding space.
Frozen space should meet national associated documents regulation, has good hygienic condition, comprises clean area, ventilation, daylighting environment.
During frozen, regularly from frozen environment, taking out cell detects.The content regularly detecting comprises that cell recovery, cytoactive detect.
Dental pulp stem cell scale production process provided by the invention has been set up a large amount of amplification methods of dental pulp stem cell; and set up the frozen system of stdn, systematize of dental pulp stem cell; set up the cell bank that long-term high quality stores dental pulp stem cell; so that for clinical and scientific research provide enough high-quality stem cell resources; with low cost, have a extensive future.
The a large amount of culturing stem cells of bio-reactor Microcarrier Culture Techniques in dental pulp stem cell scale production process of the present invention, are especially used.Compared with traditional cultural method, method of the present invention has the following advantages:
(1) microcarrier surface area/volume ratio is large, and the productive rate of unit volume culturing cell is high.More traditional monolayer cell culture area increases greatly, can obtain in a large number in a short time dental pulp stem cell;
(2) microcarrier suspension is in substratum, and dental pulp stem cell growing environment homogeneous, has simplified monitoring and the control of culture condition, and substratum utilization ratio is high simultaneously;
(3) sampling repeatability is good, and cell finally gathers in the crops that process is uncomplicated, and labour intensity is little, takes up room little, easy and simple to handle, and required personnel are few, and technique is easily amplified production, can ensure cell quality;
(4) cell culture system meets up-to-date statutory standard, and security is better.
Meanwhile, stdn, systematized dental pulp stem cell Long-term Cryopreservation method in dental pulp stem cell scale production process of the present invention, have especially been adopted.Stem cell by external extensive amplification after, frozen according to stdn flow process, frozen space has good hygienic condition, comprises clean area, ventilates, daylighting environment, meets national associated documents regulation.Meanwhile, the cell information of record is prepared against following scientific research and clinical use in detail.
Dental pulp stem cell scale production process of the present invention can not only increase the quantity of dental pulp stem cell as seed cell greatly; and can keep for a long time the ability of its Proliferation and differentiation; after Cryopreservation, also can keep the fundamental characteristics of its stem cell, after freezing preservation, can provide a large amount of dental pulp stem cell resources for the clinical and scientific research in future.
Brief description of the drawings
Fig. 1 is the schema of dental pulp stem cell scale production process of the present invention.
Embodiment
The present invention is further elaborated below to use experimental example.The art those of ordinary skill will be appreciated that, these experimental examples only do not form any restriction to scope of the present invention for illustrating the present invention, any change made for the present invention, as long as without prejudice to spirit of the present invention, all will drop within the scope of claim.
Operation in example is all carried out under strict gnotobasis, not contaminated for ensureing cell, in the phosphate buffered saline buffer of all uses, cell culture fluid, is all added with 100U/ml penicillin and 100U/ml Streptomycin sulphate.
Embodiment 1, dental pulp stem cell separation and Extraction
Fresh donor dental tissue is adopted containing antibiotic PBS solution and completes transfer, under aseptic condition, pick out pulp tissue and use PBS damping fluid to rinse; The pulp tissue picking out is shredded, add 0.3% NTx enzyme of 10 times of volumes, dispel evenly with suction pipe, be placed in 37 DEG C of shaking culture case shaking culture 120min; Complete after digestion, in suspension, add the α-MEM nutrient solution containing 15% foetal calf serum, stop digestion, at the centrifugal 5min of 1000r/min, abandon after supernatant, add the α-MEM nutrient solution re-suspended cell containing 15% foetal calf serum of 5 times of volumes, proceed in 6 orifice plates and carry out, in 37 DEG C, 5%CO
2under condition, cultivate, within 3 days, change liquid.
The two-dimentional culture vessel of embodiment 2, dental pulp stem cell is cultivated amplification (two-dimentional culture vessel comprises cell cultures orifice plate, culture dish, tissue culture square vase), and what the present embodiment adopted is that plate is cultivated.
When cell cover with plate 80% time, discard original fluid, with PBS washing 2 times, add and cover 0.25% trypsinase that contains EDTA of measuring at the bottom of ware, digestion 3-4min, uninterruptedly observes with inverted microscope during this time, see kytoplasm retraction, intercellular substance increases, cell rounding, add immediately with the isopyknic cell culture medium of pancreatin and stop digestion, repeatedly blow and beat attached cell with suction pipe, cell is blown and beaten, the centrifugal 5min of 1000r/min, using cell culture fluid re-suspended cell, is 6 × 10 according to density again
4individual cell/ml, is inoculated in plate, adds fresh cell culture fluid in 37 DEG C, 5%CO
2under condition, cultivate, within 3 days, change liquid.Can obtain 2 × 10
5individual cell/ml cell, 3 times of cell quantity amplifications.
Embodiment 3, dental pulp stem cell are containing cultivating amplification in the rolling bottle of microcarrier
What use in the present embodiment is the spinner culture amplifying cells that contains microcarrier, and rolling bottle working volume is 125ml, and microcarrier is Cytodex-3.
1. rolling bottle pre-treatment: 125ml rolling bottle cleans up, dries to complete drying, and 20ml adds silication liquid (Sigmacote, Sigma), in rolling bottle, slow circumvolve rolling bottle makes silication immersion profit bottle wall, sucks after silication liquid, rolling bottle is placed in to the air-dry 12h in ventilation, and distilled water flushing is for subsequent use;
2. microcarrier pre-treatment: in the present embodiment, microcarrier density is decided to be 2g/L, because volume of culture is 50ml, therefore take in 0.1gCytodex-3 rolling bottle, add 20ml PBS to soak 3h, after sucking, add the PBS that 20ml is new, 121 DEG C of high pressure steam sterilization 20min, suck PBS and add 20ml to contain α-MEM nutrient solution of 15% foetal calf serum, 37 DEG C of soaked overnight;
3. the inoculation of dental pulp stem cell: get 4 plates and grow to the dental pulp stem cell of 80% fusion, after 0.25% pancreas enzyme-EDTA digestion, stop digestion with the nutrient solution of 15% foetal calf serum, the centrifugal 5min of 1000rpm, fresh medium is resuspended, with 4 × 10
4the density of individual cell/ml is seeded on three-dimensional microcarrier.To contain 8 × 10
6the suspension of individual cell adds in the rolling bottle that contains Cytodex-3, then supplies respectively nutrient solution to 25ml, is placed in intermittent stirring on Wheaton magnetic stirring apparatus, and intermittent stirring condition is that 45rpm stirs 2min, stops 13min, the 3h that so circulates, 37 DEG C, 5%CO
2.After inoculation finishes, fill into respectively 25ml nutrient solution to the final working volume of 50ml, adjustment of rotational speed is 55rpm, within every 2 days, changes 2/3 nutrient solution until cultivate end.
4. obtaining of dental pulp stem cell: stop the stirring in rolling bottle, abandoning supernatant, after 0.25% pancreas enzyme-EDTA digestion, stops digestion with the nutrient solution of 15% foetal calf serum, the centrifugal 5min of 1000rpm, fresh medium is resuspended, can obtain 5.8 × 10
5the cell of individual cell/ml density, 14.5 times of cell quantity amplifications.
Embodiment 4, stirring type bioreactor are cultivated amplification dental pulp stem cell
Cultivate on a small scale amplification dental pulp stem cell with plate or rolling bottle, reach enough cell quantities.
Ligation device gas circuit pipe, liquid-inlet pipe, the pipelines such as drain pipe, 121 DEG C of high pressure moist heat sterilization 40min, squeeze into fresh cell culture fluid, stir and within 2 days, carry out nutrient solution inspection bacterium.
The hydration process of microcarrier: take 15g microcarrier and put a clean container, use without Ca
2+, Mg
2+pBS at room temperature aquation spend the night and mild stirring frequently.Supernatant discarded, adds fresh in Ca
2+, Mg
2+pBS, after mild stirring, wash 2 times, discard the PBS of washing, add PBS, 121 DEG C of high pressure moist heat sterilization 20min.
The microcarrier of prehydration, sterilizing is inoculated in 3L nutrient solution.
Select form health, well-grown plate is produced trypsinase/EDTA Digestive system digestion for cell, according to 6 × 10
5individual/mL makes cell suspension, and half amount is seeded in reactor, regulates the temperature of reactor, nutrient solution pH, and the parameters such as dissolved oxygen, are that Growth of Cells carries out cell cultures in optimum environment.80rpm turns 3min, leaves standstill 12min, so repeats 12 circulations, finishes rear inoculation remaining cell, with the rotating speed cultured continuously of 100rpm.
Adopt fill-up mode to cultivate, dissolved oxygen is the important parameter in cellular metabolism, it can affect the productive rate of cell, regulate dissolved oxygen amount to maintain 50% so need to observe constantly, this process can be controlled and add the flow of air, oxygen, nitrogen and four kinds of gases of carbonic acid gas in animal cell culture tank to make it keep best ratio to realize quantitatively in order by computer.
Computer is controlled at temperature of reactor 37 ± 0.2 DEG C in real time automatically, pH:7.0-7.2.
Cell inoculation the 2nd day, converges situation according to the growth of cell and judges whether to carry out perfusion culture and perfusion volume, and generally pouring into volume is 1 working volume of reactor, can be controlled in afterwards 1-2vvD.
Sampling every day is observed and is taken pictures, and calculates cell number.When cell covers with microcarrier surface, number reaches approximately 5.4 × 10
6individual/when mL, to stop cultivating.
Microcarrier/cell retention device by reactor is discharged highly pressurised liquid, adds 500mLPBS to rinse 2-3 time, and 100mL pancreatin/EDTA adds jar, and digestion 20min adds equal-volume nutrient solution to stop digestion, harvested cell, 9 times of cell quantity amplifications.
Embodiment 5, fixed-bed type bioreactor culture amplification dental pulp stem cell
1. adopt (the Old Hickory of Reemay company of U.S. BBA Fiberweb, TN, USA) the non-woven fabrics polyester fiber (Non Woven Polyester Fabric) of producing, No.2214 type, through surface treatment and cut into the circular shaped patches body that diameter is 6mm.
What 2. bioreactor was selected is to buy from the 5L bio-reactor Celligen of NBS company of U.S. Plus, and working volume 3.5L adopts the basket stirring system of fixed bed.The round polyester that the diameter that adds above-mentioned processing well cutting in bio-reactor is 6mm is cut into slices as cell carrier, and adds phosphoric acid buffer, 121 DEG C of sterilizing 30min.After sterilizing, emptying phosphoric acid buffer, adds the α-MEM nutrient solution containing 15% foetal calf serum, and starts to control bioreactor condition.
3. select form health, well-grown trypsinase/EDTA Digestive system digestion for dental pulp stem cell, as the seed cell of bioreactor, with 3 × 10
5the density of individual cell/mL is inoculated on the non-woven fabrics/trevira circular shaped patches carrier in bio-reactor, carries out amplification cultivation with cell culture fluid.Wherein the usage quantity of trevira circular shaped patches carrier is every liter of tank volume 35g, and the cell culture fluid using adds 15% foetal calf serum for α-MEM substratum.Bioreactor culture condition is potential of hydrogen (pH) value 7.2,37 DEG C of temperature, oxyty (DO) for air saturation 50%, stirring velocity 80rpm.
4. detect the parameter such as glucose and lactic acid content by Biochemical Analyzer every day, after inoculation, cultivate approximately 8 days emptying nutrient solution, add 500mLPBS to rinse after 2-3 time, add 100mL pancreatin/EDTA, digestion 20min, add equal-volume nutrient solution to stop digestion, harvested cell, can obtain 3.8 × 10
6the cell of individual cell/ml, 12.6 times of cell quantity amplifications.
Embodiment 6, WAVE bio-reactor
1. adopt the WAVE Bioreactor system of GE, it is made up of a shake platform with disposable plastic cell culture bags, is equipped with CO
2gas mixer (CO2MIX20) or WAVEPOD
tMcontrol tower is used for controlling pH, temperature, oxygen and mixing.Dental pulp stem cell is cultivated in the bio-reactor of Cellbag-2L.
2. microcarrier pre-treatment: using not containing Ca
2+and Mg
2+in the PBS of ion, wash after 3 times, microcarrier is at 121 DEG C of autoclaving 15min, and at 4 DEG C Preservation in sterile condition.Microcarrier first with substratum washing and before inoculation 24h transfer to and in Cellbag, carry out balance.Cytodex 3 amounts that use are 3g/l.
3. select form health, well-grown 0.25% trypsinase for dental pulp stem cell/EDTA digestion, as the seed cell of bioreactor, with 2 × 10
5the density of individual cell/ml is inoculated on microcarrier, carries out amplification cultivation with cell culture fluid.The cell culture fluid using adds 15% foetal calf serum for α-MEM substratum.Bioreactor culture condition is potential of hydrogen (pH) value 7.2,37 DEG C of temperature, oxyty (DO) for air saturation 50%.
4. detect the parameter such as glucose and lactic acid content by Biochemical Analyzer every day, after inoculation, cultivate approximately 7 days emptying nutrient solution, add 500mLPBS to rinse after 2-3 time, add 100mL pancreatin/EDTA, digestion 20min, add equal-volume nutrient solution to stop digestion, harvested cell.Can obtain 2.2 × 10
6the cell of individual cell/ml density, 11 times of cell quantity amplifications.
The freezing preservation of embodiment 7, dental pulp stem cell
By the dental pulp stem cell covering with in plate to 80-90%, remove old nutrient solution, with PBS cleaning 2 times, then add 37 DEG C of digestion 3min of 0.25% pancreatin that contains EDTA, after cell all comes off, add isopyknic dental pulp stem cell nutrient solution to stop digestion, the centrifugal 5min of 1000rpm/min, remove supernatant, use again stem cell cryopreserving liquid (to contain DMSO, foetal calf serum and α-MEM nutrient solution, 1:2:7) that cell is resuspended, the dental pulp stem cell suspension obtaining joins in cell cryopreservation tube, be placed in containing program mode cooling box-80 DEG C of Virahol and spend the night, within second day, transfer to the medium-term and long-term preservation of liquid nitrogen of-196 DEG C.
Claims (10)
1. a scale production process for dental pulp stem cell, comprises the steps:
(1) separation and Extraction of dental pulp stem cell: by pulp tissue with after collagenase digesting, containing in the culture vessel of foetal calf serum cell culture fluid, in 37 DEG C, 5%CO
2under condition, cultivate, within 3 days, change liquid;
(2) cultivation of dental pulp stem cell amplification: the primary cell that step (1) is cultivated to gained is with 0.1 × 10
5-1.0 × 10
5individual cell/cm
2density be inoculated in two-dimentional culture vessel, add fresh cell culture fluid, in 37 DEG C, 5%CO
2under condition, cultivate, within 3 days, change liquid;
(3) detection of dental pulp stem cell: carry out cytoactive detection, sterility detection and fluidic cell and detect;
(4) large scale culturing of dental pulp stem cell: adopt spinner culture or bioreactor culture, after dental pulp stem cell is seeded in rolling bottle or bio-reactor, adjust dissolved oxygen concentration, pH value, mixing speed and air flow, change substratum according to the result of sampling analysis cell counting, survival rate, nutritive ingredient and metabolite, thereby reach the high-density large-scale cultivation of cell;
(5) the freezing preservation of dental pulp stem cell: will detect qualified cell cryopreservation in pipe, the details of cell are scanned into data management system, automation system is according to liquid nitrogen situation Automatic-searching vacant locations, by cell cryopreservation in corresponding space;
(6) as required, carry out the long-term frozen of dental pulp stem cell and preserve, between preservation period, regularly from frozen environment, take out cell and detect.
2. production technique as claimed in claim 1, in wherein said step (1), described pulp tissue selects under aseptic condition, after rinsing, shreds with damping fluid; Described collagenase digesting is 0.1% ~ 0.6% collagenase with 5 ~ 10 times of volumes, shaking culture 30 ~ 180min in 37 DEG C of incubators, then stop digestion with the cell culture fluid containing foetal calf serum, described cell culture fluid is selected from commercial α-MEM, DMEM substratum and serum free medium containing 10-20% foetal calf serum.
3. production technique as claimed in claim 1, the culturing step of wherein said step (1) comprises and will after the centrifugal 3 ~ 10min of 800 ~ 1500r/min, abandon supernatant, add 5 ~ 10 times of volume cell culture fluid re-suspended cells through the pulp tissue of digestion, proceed in culture vessel and cultivate, described cell culture fluid is selected from commercial α-MEM, DMEM substratum and serum free medium containing 10-20% foetal calf serum.
4. production technique as claimed in claim 1, in wherein said step (2), described primary cell digests with pancreatin solution and blows and beats cell in the time covering with culture vessel bottom 80%-90%, after making cell depart from primary culture vessel completely, be inoculated in two-dimentional culture vessel, described two-dimentional culture vessel is selected from cell cultures orifice plate, culture dish, tissue culture square vase.
5. production technique as claimed in claim 1, in wherein said step (4), described dissolved oxygen concentration is that 40%-60%, pH value are that 7.0-7.2, mixing speed are 50-100rpm.
6. production technique as claimed in claim 1, described spinner culture in wherein said step (4) is undertaken by following process: by microcarrier after pre-treatment, rolling bottle silication, 121 DEG C of high-temperature sterilizations, inoculating cell, microcarrier add-on is 2-10g/L, magnitude range is 60 ~ 300 μ m, and inoculum density is 3 × 10
4-3 × 10
5individual cell/ml, the magnitude range of rolling bottle is 25ml-500ml, speed range 50-75rpm.
7. production technique as claimed in claim 1, the described bio-reactor in wherein said step (4) is selected from biological reactor for cell culture, comprises stirring type bioreactor, fixed-bed bioreactor and WAVE bio-reactor.
8. production technique as claimed in claim 7, wherein said stirring type bioreactor, WAVE bio-reactor carry out cell cultures by following process: microcarrier PBS is rinsed 2 times, adding for the third time PBS to put into 4 DEG C of refrigerator inner equilibrium aquations spends the night, after high pressure steam sterilization, inoculation dental pulp stem cell, the add-on of microcarrier is 3-10g/L, and inoculum density is 1 × 10
5-10
6individual cell/mL, cell amplification to density is 5 × 10
5-2 × 10
7individual cell/mL; Wherein said fixed-bed bioreactor carries out cell cultures by following process: in bio-reactor, add polyester slice through surface treatment well cutting as cell carrier, and add phosphoric acid buffer, after high-temp steam sterilizing, add cell culture fluid, inoculation dental pulp stem cell, wherein the usage quantity of trevira chip carrier is every liter of tank volume 25-45g, and inoculum density is 1 × 10
5-10
6individual cell/mL, cell amplification to density is 4 × 10
6-2 × 10
7individual cell/mL.Described in above-mentioned bioreactor culture condition, dissolved oxygen concentration is that 40%-60%, pH value are that 7.0-7.2, mixing speed are 50-100rpm.
9. production technique as claimed in claim 1, in wherein said step (5), the details of described cell comprise donor name, cell algebraically, cell culture fluid composition and concentration, cell inoculum density, frozen storing liquid composition and frozen date; Described freezing preservation comprises obtained dental pulp stem cell by 1 × 10
6~ 1 × 10
7individual/ml cell density is resuspended in frozen storing liquid, is distributed into cryopreservation tube, after programmed cooling, cryopreservation tube is transferred to freezing preservation in-196 DEG C of liquid nitrogen.
10. production technique as claimed in claim 1, the frozen storing liquid that the freezing preservation of wherein said dental pulp stem cell is used comprises DMSO, foetal calf serum and the α-MEM nutrient solution that volume ratio is 1:2:7.
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| CN104694464A (en) * | 2015-02-09 | 2015-06-10 | 安徽新生命干细胞科技有限公司 | Clinical dental pulp stem cell and preparation method thereof |
| CN104705289A (en) * | 2015-03-01 | 2015-06-17 | 安徽新生命干细胞科技有限公司 | In-vitro tooth preservation liquid |
| CN104711219A (en) * | 2015-03-01 | 2015-06-17 | 安徽新生命干细胞科技有限公司 | Dental pulp stem cell culture medium |
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| CN106011053A (en) * | 2016-06-27 | 2016-10-12 | 安徽新生命干细胞科技有限公司 | Novel dental pulp stem cell culturing medium |
| CN106377799A (en) * | 2016-10-13 | 2017-02-08 | 南通大学附属医院 | Preparation method of dental pulp stem cell and chitosan scaffold complex |
| CN112469815A (en) * | 2018-07-31 | 2021-03-09 | Jcr制药股份有限公司 | Method for producing dental pulp-derived cells |
| WO2020027163A1 (en) * | 2018-07-31 | 2020-02-06 | Ｊｃｒファーマ株式会社 | Method for producing dental pulp-derived cells |
| JPWO2020027163A1 (en) * | 2018-07-31 | 2021-09-24 | Ｊｃｒファーマ株式会社 | Method for producing dental pulp-derived cells |
| IL280387B1 (en) * | 2018-07-31 | 2024-03-01 | Japan Chem Res | Method for producing dental pulp-derived cells |
| CN108998344A (en) * | 2018-08-06 | 2018-12-14 | 武汉赛科成科技有限公司 | A kind of method of continuous large-scale production recombined adhenovirus |
| CN108998344B (en) * | 2018-08-06 | 2022-07-05 | 武汉赛科成科技有限公司 | Method for continuous large-scale production of recombinant adenovirus |
| CN110004114A (en) * | 2019-04-02 | 2019-07-12 | 浙江优牙生物科技有限公司 | A kind of serum free medium of dental pulp stem cell |
| CN110951683A (en) * | 2020-01-06 | 2020-04-03 | 深圳华云生物科技发展有限公司 | Preparation method of dental pulp stem cells |
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