CN101993853B - Injection type vascularized adipose tissue and construction method thereof - Google Patents
Injection type vascularized adipose tissue and construction method thereof Download PDFInfo
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- CN101993853B CN101993853B CN200910090507XA CN200910090507A CN101993853B CN 101993853 B CN101993853 B CN 101993853B CN 200910090507X A CN200910090507X A CN 200910090507XA CN 200910090507 A CN200910090507 A CN 200910090507A CN 101993853 B CN101993853 B CN 101993853B
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Abstract
The invention relates to a method for constructing a blood vessel, in particular to an injection type vascularized adipose tissue and a construction method thereof. In the invention, autologous cells are used for simulating a natural vascular tissue, and a natural biological material is used for packaging tissue cells to form microballoons; smooth muscle cells and endothelial cells are compounded to form a blood vessel structure by utilizing gaps of the microballoons as blood vessel forming channels to obtain an artificial tissue with a blood vessel structure; by simulating the natural vascular tissue, adipose stem cells are packaged into the natural material to prepare the microballoons; and a vascularized factor slow release system is combined to construct the injection type vascularized adipose tissue. According to the invention, the injection type vascularized adipose tissue can be constructed in vitro or in vivo and used for repairing small and large soft tissues, can stably exist in the human body for a long time, has small operational injury and quick postoperative recovery and also can be used for an in vitro biochemically or pharmacally researched model. The method for constructing the blood vessel and the artificial tissue also can be used for other artificial tissues, such as liver tissues, cartilaginous tissues, and the like.
Description
Technical field
The present invention relates to bioengineering field, particularly relate to a kind of injection type vascularized adipose tissue and construction process thereof based on autologous stem cells.
Background technology
There are millions of routine patients in the annual whole world, because the soft tissue that wound, tumorectomy and birth defects cause, face and Mastectomy need to carry out reconstruction by operation.Because mammary cancer is one of modal malignant tumour of women, sickness rate is up to 10%～15%, and is and rises year by year and the trend of rejuvenation, and the soft tissue that tumor resection causes and Mastectomy quantity are maximum.According to U.S. orthopedist association, within 2004, the whole world has 5,000,000 people of surpassing to carry out the breasst reconstruction operation, wherein has 4,000,000 to be due to tumor resection.
It is generally acknowledged, desirable fatty soft tissue filling material should have similar flexibility, identical osmotic pressure, stable physicochemical property, good biocompatibility, not carcinogenic, not sensitization, not teratogenesis, not calcification etc. with natural tissues.The breast more generally used at present and face are filled and are reproduced material and comprise medical material and the large class of autograft two.Medical material comprises again embedded type prosthese and the injection mixtures such as whiteruss, Vaseline such as silica gel, polyacrylamide gel, triacylglycerol (triglyceride level), hyaluronic acid.Current research shows to use these materials to be prone to the complication such as prosthesis ruptures or seepage, inflammatory reaction, granuloma, capsule contracture, prostheses migration, infection, mondor's disease, and security is subject to very large query.Take silicone as example, and FDA in 1992 have been forbidden the use of all silicones.
Comparatively speaking, it is abundant that autograft has source, have wide range of applications, and good biocompatibility, without immunological rejection, good hand touch, many advantages such as safe and reliable and capable of regulating.For the damaged and plasticity that reaches among a small circle small volume, Autofat granule injection is a kind of scheme of clinical frequent use at present.The method is usingd fat granules as the tissue packing material, without rejection, does not have pathogenic, carinogenicity.The position that source of drawing material is more in body fat, can carry out liposuction procedures in filling, obtains the effect that many benefits are killed two birds with one stone less simultaneously.For staying hardly the operation scar in He Shou district, district, on normal anatomical structures impact and secondary all very little, secure persistent of damaging of human body.Indication is extensive, and almost human epidermal undertissue depression is damaged all can fill.From Van der Meulen reported first in 1889 since the autotransplantation of fatty tissue, this technology successfully is applied to that 1. Facial Depression is damaged or lopsided, as cheekbone section, temples, forehead, socket of the eye; 2. darker wrinkle, nasolabial groove and saddle nose; 3. upper lip is excessively thin, and ear-lobe is less etc.; 4. breast dysplasia, the atrophy of lactation rear udder attachment, the asymmetric and crater nipple of bilateral breast; 5. penis increases thick, rich labium majus [pudendi, slack vagina deflation, the back of the hand soft tissue atrophy filling.This treatment plan shortcoming is can not set up well blood supply system owing to being injected into intramammary fat particle, when significant quantities of fat is accumulated in together, can, because blood supply insufficiency causes the adipocyte necrosis, dissolves, absorbs, very easily cause the sequela such as infection, hemotoncus, distortion, pain, scleroma, tumour, necrosis.Research finds approximately to have 50%～60% fat particle to be absorbed, and what survive organizes also gradually by fibrosis or calcification, and effect is not lasting, often needs multiple injection to maintain shape.
Soft tissue defects for large volume, especially the reparation after lumpectomy, the operative treatment schemes of lower abdominal flap are shifted in many employings at present, transplant free musculo cutaneous flap to reach the mamaplasty purpose by the micro-vascular surgery anastomosis, its profile is true to nature, of good quality, be easy to moulding, blood fortune system is good, the Dan Geigong district inevitably causes wound and scar, cause dysfunction, as local muscle neuratorphy, belly weakness and formation abdominal hernia etc., and after transplanting, absorb obviously, effect can not be lasting, the untoward reactions such as transplanted tissue's necrosis especially can appear when the torsion of the base of a fruit with base of a fruit tissue transplantation body or pressurized.The patient usually is difficult to accept this way, and the graft donor is subject to corresponding restriction.
At present, application organizational engineering principle builds fatty tissue has become one of the focus in Tissue Engineering Study field.
Aspect seed cell research, due to the adult adipocyte (individual large, wall is thin, in fat particle is arranged) not proliferative and subject to damage, people more and more turn to interest and have specific differentiation potential, the stem cell of good self and amplification ability.Being proved can be to adipocyte, chondrocyte, myocardial cell for fat stem cell (adipose derived adult stem cells), and the multiple directions such as neurocyte and scleroblast are directed to be transformed.And that autologous fat stem cell has the adipose tissue-derived that can Gong draw materials is abundant, the advantage such as it is convenient to extract, and can repeatedly obtain, and the operation damage is little, and without immune rejection, cell is easily cultivated, and rate of propagation is very fast, accepted by increasing people; In addition, front adipocyte is a kind of mechanocyte, can breed also specificity and, to Adipocyte Differentiation, can separate from the fatty tissue through enzymic digestion or subcutaneous lipids aspirate, and the resistibility of physical abuse is stronger to external world.Just can grow with common cell culture processes, and can increase in vitro and break up, the research of adipose tissue engineering at present also mainly concentrates on fat stem cell and front adipocyte.
Aspect timbering material, the polyglycolic acid in the aliphatic polyester series biodegradable polymer (polyglyclic aicd), poly(lactic acid) (polylactic acid) and both multipolymers (polylacticacid/polyglyclic aicd copolymers), collagen etc. are for experimentation on animals.But exist subject matter to be: synthesized polymer material commonly used seems too hard with respect to breast tissue on the one hand, if can not set up in time the blood confession on the other hand, the degradable high polymer material of compound cells can be absorbed by body equally, is difficult to stable for extended periods of time.The same with the injection-type fat particle, material and cell are degraded, absorb, and the unsettled principal element of three-dimensional structure remains can't set up the blood supply deficiency that vasoganglion causes in time.
The existing research that builds vasoganglion with microsphere system at present, research mainly concentrates on the reserving space as vascularization with the passage formed after synthetic materials microballoon sintering, then compound endotheliocyte, it is respond well, but compares with fatty tissue or very large distance is arranged.
In sum, autotransplantation is all current treatment plan more commonly used for small volume and the damaged reparation of large volume, and wherein the injecting type fat particle can be accepted by the patient, but has the unsurmountable problems such as drainage area wound and absorption.Adipose tissue engineering provides a kind of very promising direction in conjunction with self stem cell technology, but the current also progress of not making a breakthrough property, maximum difficult point is graft degraded, the absorption or downright bad that blood supply insufficiency causes.
Summary of the invention
The purpose of this invention is to provide a kind of microvascular method of structure.
Another object of the present invention is to provide a kind of injection type vascularized adipose tissue and construction process thereof based on autologous stem cells.
The method of structure blood vessel of the present invention, be to adopt natural biologic material to make microballoon histocyte, utilizes gap between microballoon as the passage that forms blood vessel, builds blood vessel, mainly comprises the following steps:
1) autologous stem cells is induced respectively into smooth muscle cell and endotheliocyte, smooth muscle cell evenly mixes with coating material, then add the histocyte microballoon, make it can form at least two-layer in culture space, cultivated, until the coating material that contains smooth muscle cell is evenly distributed on the histocyte microsphere surface and by the self-assembly effect, microballoon is connected into to integral body, the gap between microballoon forms passage;
2) at the compound endotheliocyte of channel inner surface.
The method of structure blood vessel of the present invention, in step 1) before, also comprise and build the histocyte microballoon: histocyte is wrapped in sodium alginate based aquagel material, makes the histocyte microballoon;
In step 3) afterwards, also comprise step 4) the vascularization somatomedin is wrapped in sodium alginate based aquagel material, make the cytokine sustained-release micro-spheres, then with step 3) homogeneous microstructure that obtains mixes.
Step 1) described histocyte microballoon and step 4) described cytokine sustained-release micro-spheres can adopt the whole bag of tricks that histocyte or somatomedin are wrapped in sodium alginate based aquagel material and make microballoon, preferred non-contact type high voltage electrostatic method, process is as follows: by histocyte or somatomedin with 10
6～10
7extrude by syringe pump after the density of individual/ml and sodium alginate based aquagel material mixing, splash in solid solution through the pulling force of high tension electrostatic field, make histocyte or somatomedin microballoon;
Wherein, described sodium alginate based aquagel material can be one or more in sodium alginate, sodium alginate/collagen, sodium alginate/glutin, sodium alginate/Fibrinogen; Described solid solution is the salts solution containing divalent cation, as calcium chloride, bariumchloride etc., and preferably calcium chloride solution.
In described sodium alginate base biomaterial, the ultimate density of sodium alginate is 1-10%, and gelatin, collagen or fibrinogenic ultimate density are 0.01-5%, and calcium chloride solution concentration is 100mM～500mM.
The particle diameter of described histocyte microballoon or somatomedin microballoon can decide according to those skilled in the art's needs, preferred micron order, and for ease of coordinating, the particle diameter of somatomedin microballoon should be less than the particle diameter of histocyte microballoon; The particle diameter of preferred histocyte microballoon is 100 μ m～500 μ m; The particle diameter of cytokine sustained-release micro-spheres is 20 μ m～50 μ m.
Described coating material is the biomaterial that cellular affinity is high, the self-assembly effect is arranged, and is preferably selected from one or more of collagen, Fibrinogen, matrigel etc.
Described step 2) carry out as follows: using the endotheliocyte suspension as nutrient solution, by step 1) the tissue utilization for preparing pulsation is cultivated or common training method is cultivated, make endotheliocyte be attached to the inwall of described passage, the formation outer wall is the vascular tissue that smooth muscle cell, inwall are endotheliocyte.Described endotheliocyte suspension comprises induces the endothelialization substratum, and culture condition determines according to the known technology of this area.
In the method for structure blood vessel of the present invention, described cultivation is to carry out in certain culture space, described culture space can be selected the formed space of cultivation apparatus commonly used, various this areas, as centrifuge tube, 12 orifice plates etc., preferably, culture space should be able to make microballoon form at least two-layer at least one direction, thereby make can form between two-layer microballoon the through channel of certain-length, then cultivate, until the coating material that contains smooth muscle cell is evenly distributed on the histocyte microsphere surface and by the self-assembly effect, microballoon is connected into to integral body, through channel between the gap between microballoon and two-layer microballoon forms passage, finally with the endotheliocyte suspension, as nutrient solution, pulse and cultivate or common cultivation, build blood vessel.
The present invention also provides a kind of injection type vascularized adipose tissue based on autologous stem cells.
Injection type vascularized adipose tissue of the present invention is the composition and structure that imitates the natural fat tissue, adopt autogenous cell fully, form the micron order bead with natural biologic material parcel cell, utilize Interglobular hole as angiopoietic passage, and, in conjunction with vascularization factor slow release system, construct the Vascularized fat depot of injection type.Comprise the following steps:
1) structure of adipocyte microballoon: get fat stem cell, be wrapped in sodium alginate based aquagel material and form the fat stem cell microballoon, then, with becoming fat inducing culture liquid to cultivate, make fat stem cell be divided into adipocyte, obtain the adipocyte microballoon;
2) build blood vessel with above-mentioned method, wherein histocyte is fat stem cell, comprising: i) fat stem cell is induced into respectively to smooth muscle cell and endotheliocyte; Ii) smooth muscle cell is evenly mixed with coating material; Iii) will add the adipocyte microballoon, with the coating material that comprises smooth muscle cell, evenly mix, make the adipocyte microballoon can form at least two-layer in culture space, cultivated, until the coating material that contains smooth muscle cell is evenly distributed on the adipocyte microsphere surface and by the self-assembly effect, the adipocyte microballoon is connected into to integral body, the gap between microballoon forms as the passage that builds microvascular space; Iv), at the compound endotheliocyte of channel inner surface, form blood vessel/adipocyte system;
3) structure of cytokine sustained-release micro-spheres: the vascularization cytokine is wrapped in sodium alginate based aquagel material, makes the cytokine sustained-release micro-spheres;
4) the cytokine sustained-release micro-spheres and the step 2 that the structure of injection type vascularized adipose tissue: by step 3) make) blood vessel/adipocyte system that obtains mixes, and makes injection type vascularized adipose tissue.
Wherein, step 1) described fat stem cell is autogenous cell, can obtain by variety of way well known in the art, and special, the autologous fat that can adopt liposuction to obtain organizes to extract fat stem cell, and this leaching process is techniques well known; And in addition, the fat stem cell that extraction can be obtained first carries out external plane and cultivates amplification, obtains being used further to prepare the fat stem cell microballoon after desired number;
Described fat stem cell microballoon can adopt the whole bag of tricks well known in the art preparation, preferred non-contact type high voltage electrostatic method, its preparation process is as follows: by fat stem cell with 10
6～10
7extrude by syringe pump after the density of individual/ml and sodium alginate based aquagel material mixing, splash in solid solution through the pulling force of high tension electrostatic field, make the stem cell microballoon; Equally, the cytokine sustained-release micro-spheres step 3) also can adopt same method to carry out: the vascularization somatomedin is made with splashing in solid solution after sodium alginate mixes.
Wherein sodium alginate based aquagel material can be one or more in sodium alginate, sodium alginate/collagen, sodium alginate/glutin, sodium alginate/fibronectin; Described solid solution is the salts solution containing divalent cation, as calcium chloride, bariumchloride etc., and preferably calcium chloride solution.
In described sodium alginate based aquagel material, the ultimate density of sodium alginate is 1-10%, and gelatin, collagen or fibrinogenic ultimate density are 0.01-5%, and calcium chloride solution concentration is 100mM～500mM.
The particle diameter of described fat stem cell microballoon is 100 μ m～500 μ m; The particle diameter of described cytokine sustained-release micro-spheres is 20 μ m～50 μ m.
Described vascularization cytokine is for promoting capillary vessel that the cytokine of mature blood vessel occurs and promotes, comprise the blood vessel relative growth factors such as vascular endothelial growth factor (VEGF), Prostatropin (bFGF), Urogastron (EGF, Epidermal Growth Factor), Thr6 PDGF BB (PDGF), transforming growth factor (TGF-β 1) and Ang-1 (angiopoietin-1).
Step 1) or step 2) described in fat stem cell is induced to lipoblast, smooth muscle cell and endotheliocyte are methods well known in the art, and fat stem cell is cultivated and got final product under the effect of corresponding inductor.
Step 1) the fat stem cell microballoon in is after becoming fat to cultivate, and fat stem cell wherein, owing to being divided into adipocyte, will be full of high density adipocyte cluster in microballoon; One-tenth fat inductor well known in the art is comprised of multiple, as: DMEM in high glucose, 10% foetal calf serum, 0.5mmol/L IBMX, 1 μ mol/L dexamethasone, 10 μ mol/L Regular Insulin, 1% mycillin stoste etc., all can be used for the present invention;
The endothelium inductor of fat stem cell being induced into to endotheliocyte also is comprised of multiple, as low sugar DMEM, and 2% foetal calf serum, 2% horse serum, 10ng/mL VEGF, 2ng/mL bFGF (R& D system), 1 * ITS (Sigma), 0.61g/L nicotinamide, 0.1 μ mol/L dexamethasone, 2g/L BSA, 0.1g/L ornithine, 0.03g/L proline(Pro), 0.73g/L glutamine, 1g/L glucose, 2g/L semi-lactosi and appropriate trace element (zinc chloride, zinc sulfate and Manganous chloride tetrahydrate) etc.; The unstriated muscle inductor of fat stem cell being induced into to smooth muscle cell also is comprised of multiple, for example: containing the M-199 induced liquid of 5ng/mL TGF-β 1 and 50ng/mLPDGF-BB.
Step 2) described coating material is the biomaterial with high cellular affinity of self-assembly characteristic, as one or more in the biomaterials such as collagen, Fibrinogen, matrigel; Preferred 0.01mg/ml～the 100mg/ml of its working concentration;
Wherein, step I ii) described cultivation is the coating material that will comprise smooth muscle cell with after the adipocyte microballoon evenly mixes, standing cultivation in certain culture space, until coating material fully is combined with microsphere surface and the self-assembly effect is occurred mutually, then cellar culture or pulsation are cultivated until smooth muscle cell attaches on coating material and sprawls growth; The nutrient solution now adopted is the corresponding nutrient solution of inducing smooth muscle cell, and composition has and enumerates in front in detail, is known technology; Culture condition is the condition of conventional cultivation smooth muscle cell.Step I ii) described incubation time is preferably 24～72 hours.
As nothing specializes, the cultivation that this paper mentions is all to cultivate in CO2gas incubator except pulsation is cultivated.
Described step I v) described in, the method for compound endotheliocyte is: using the endotheliocyte suspension as nutrient solution, by step I ii) the tissue utilization for preparing pulsation is cultivated or common training method is cultivated, make endotheliocyte be attached to the inwall of passage, the formation outer wall is the vascular tissue that smooth muscle cell, inwall are endotheliocyte.The substratum adopted and culture condition are that this area inducing endothelial cell is commonly used, by those skilled in the art, according to the known technology of this area, are determined.
Centrifugal rear evenly being suspended from in endothelium inducing culture liquid of endotheliocyte digestion of specifically, plane being cultivated; The adipocyte microballoon that has mixed coating material is evenly mixed with the endotheliocyte suspension, mix the just space between microballoon of rear endothelium suspension, in passage; The cultivation of conventional plane or pulsation are cultured to endotheliocyte and attach to microballoon and channel surface, and make the endotheliocyte that closes on microsphere surface couple together; The final pipeline formed by the composite structure that outwards is followed successively by endothelial layer, layer of smooth muscle cells, coating material layer, adipocyte cluster, obtain blood vessel/adipocyte system.
Described non-contact type high voltage electrostatic method prepares microballoon and can adopt the non-contact type high voltage electrostatic power unit of Chinese patent application 200910079726.8 to carry out; Described pulsation is cultivated and can be selected the pulsation incubator of Chinese patent application 200910079726.8 to carry out, and therefore, the present invention by Chinese patent application 200910079726.8 as a reference.
As mentioned above, the specific embodiment of the invention method is as follows:
Its operating process is shown in Fig. 2.Adopt liposuction to obtain patient's autologous fat stem cell, cellar culture is bred to sufficient amount; Taking out a part of fat stem cell adopts non-contact type high voltage electrostatic power unit (seeing Chinese patent application 200910079726.8) to make solid-state fat stem cell microballoon, core materials is sodium alginate based aquagel material, as one or more in sodium alginate, sodium alginate/collagen, sodium alginate/glutin, sodium alginate/Fibrinogen, solid solution is calcium chloride solution.To the fat stem cell microballoon, become fat to cultivate, formed the high density adipocyte cluster of material parcel;
To the part fat stem cell, become unstriated muscle to induce to form smooth muscle cell, by its digestion, centrifugal, be dispersed in the coating material solution such as collagen, Fibrinogen, matrigel after collecting, the density of adjusting smooth muscle cell is 10
6～10
8individual/ml, coating material concentration is 0.01mg/ml～100mg/ml; The coating material that will comprise smooth muscle cell with after the adipocyte microballoon evenly mixes in limited space standing cultivation enough time, preferably 24～72 hours, coating material fully is combined with microsphere surface and the self-assembly effect occurs mutually, cellar culture or pulsation are cultivated until smooth muscle cell attaches on coating material and sprawls growth;
To the part fat stem cell, become endothelium to induce to form endotheliocyte, by its digestion, centrifugal, be dispersed in the microsphere system that has attached smooth muscle cell after collecting, cellar culture or pulsation are cultivated until endotheliocyte attaches to smooth muscle layer and channel surface.
Described cultivation is to carry out in certain culture space, described culture space can be selected the formed space of cultivation apparatus commonly used, various this areas, as centrifuge tube, 12 orifice plates etc., preferably, it is at least two-layer that culture space should be able to make the adipocyte microballoon form at least one direction, makes can form between two-layer microballoon the through channel of certain-length; In addition, for making the injectable fatty tissue obtained, be more preferably less than the space of injection needle tubing, certainly also can adopt larger culture space to build the Vascularized fat depot of comparatively large vol, during injection, the Vascularized fat depot of structure is divided into to appropriate size and gets final product.
Adopt non-contact type high voltage electrostatic power unit vascularization cytokine processed (as bFGF, VEGF etc.) sustained-release micro-spheres.
Then, the adipocyte microsphere system that is compounded with smooth muscle cell and endotheliocyte is evenly mixed with the somatomedin microballoon, obtain injection type vascularized adipose tissue (seeing the schematic diagram of Fig. 1), can proceed subsequently cellar culture or pulsation cultivation, perhaps it is sucked in disposable syringe, inject the position of needs of patients reparation or plasticity.Due to the particle diameter of having controlled cell microballoon and somatomedin microballoon, vascularization somatomedin microballoon will be distributed in the passage of cell microsphere system.This mixture has the bionical microvessel structure built in advance, and along with the degraded of diffusion process and solid support material, somatomedin is released gradually, promotes the formation of microvascular continuation growth and maturation and capillary network.
Vascularization is important, the problem demanding prompt solution that field of tissue engineering technology generally faces.Blood vessel construction process of the present invention, the direct construction by microvessel structure and capillary vessel induce generation, can solve absorbed problem after tissue transplantation, for tissue repair provides good solution.
The construction process of the Vascularized fat depot based on autogenous cell combines cell microsphere preparation technology, growth factor slow-release technology and impulsive motion bioreactor Vitro Culture Techniques.Natural materials in microballoon provides the porous three-dimensional microenvironment for cell, can be as the physical support and the structure space that maintain the cell normal activities; There is very high physical strength, can protect adipocyte injury-free in the process of basis and shaping; Can build the high local concentrations cell cluster, form bionical cystic structures, the spatial positioning of cell is regulated and controled, can realize mixing and the compound system of various kinds of cell simultaneously, be external basis of reproducing tissue; Space between microsphere surface and microballoon provides reserving space and blood vessel access for vascularization.The growth factor slow-release system is the important means of regulating cell Growth and Differentiation and tissue development.Pulsation cultivate vitro culture to structure can provide circulation, the pulsation culture systems, promoted the vascularization process.
Vascularized fat depot of the present invention adopts autogenous cell fully, on the basis of simulation natural fat structural constituent and constructional feature, form the micron order bead with natural biologic material parcel cell, utilize Interglobular hole as angiopoietic passage, by compound autologous smooth muscle cell and endotheliocyte form blood vessel structure around high density adipocyte cluster, can form in vitro the class fatty tissue with microvessel structure.In conjunction with vascularization factor slow release system, can construct the Vascularized fat depot of injection type.
Vascularized fat depot of the present invention adopts the modus operandi of injecting type, and wound is little, and operative results is fast, can generally be accepted by the patient.Vascularization factor slow release system can be induced in vivo capillary network to generate and be promoted further growth and the maturation of Microvasculature, final along with the degraded of biomaterial and the fusion of autogenous cell and patient's body, formation is the autologous fat tissue existed steady in a long-term in vivo.According to the present invention, can obtain in vitro and in vivo the Vascularized fat depot formed by autogenous cell and degradable biomaterial, this artificial fatty tissue has adipocyte cluster and blood vessel structure, the cytokine sustained-release micro-spheres is blended in blood vessel, induces in vivo capillary network to generate and steady in a long-term the existence.
And the cell microsphere preparation technology that the present invention adopts, blood vessel constructing technology, growth factor slow-release technology and pulsation culture technique can and build the research such as its hetero-organization (as hepatic tissue, mammary tissue etc.) for the Growth and Differentiation of external various stem cells and supply a model and platform.
The accompanying drawing explanation
Fig. 1 is that the Vascularized fat depot structure builds schematic diagram.
Fig. 2 is the implementation method schema.
Fig. 3 is the injection type Vascularized fat depot under opticmicroscope; A: integral body, scale: 50 microns; B: for the oil red O stain of adipocyte, scale: 50 microns (adipocyte takes on a red color); C: microvessel structure is observed, scale: 10 microns; D: growth factor slow-release systematic observation, scale: 100 microns.
Embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.As without specializing, in embodiment, somatomedin used and reagent are all purchased from Sigma company.
The preparation of embodiment 1 adipocyte microballoon
Adopt liposuction to obtain patient's autologous fat, after rinsing fatty tissue with phosphoric acid buffer, then add 0.75g/L type i collagen enzyme, 37 ℃ of concussions, digestion 60min, foetal calf serum stops digesting.The centrifugal 10min of 1200r/min, remove the remaining tissue of supernatant liquor and suspension, and re-suspended cell adds the erythrocyte cracked liquid (NH of 2 times of volumes
4cl 154mmol/L+KHCO
310mmol/L+EDTA 0.1mmol/L) standing 10min is centrifugal, abandon supernatant liquor.With appropriate PBS liquid, clean 3 times, 200 eye mesh screens filter, and obtain Adipose Tissue.
The gained Adipose Tissue is inoculated in to 25cm
2culturing bottle, add 5mL containing the volume fraction DMEM in high glucose substratum that is 0.10 foetal calf serum, mix the CO that to be placed in 37 ℃, volume fraction be 0.05
2in the saturated humidity incubator, cultivate.Phase microscope observation of cell morphological specificity and propagation situation, change liquid first after 36～48h, later every 72h changes liquid 1 time.Until cytogamy, at the bottom of surpassing culturing bottle 80% the time, conventional trysinization is gone down to posterity.Cellar culture is bred to sufficient amount.
Get the fat stem cell that grows to incorporating period between the 4th 0 generations of generation to the, adopt sodium alginate/collagen system as becoming capsule material, with the non-contact type high voltage electrostatic power unit, make solid-state fat stem cell microballoon:
1. dissolve sodium alginate with phosphoric acid buffer, the acetic acid with 2% dissolves collagen makes solution.Both are mixed into to the NaOH solution that sodium alginate and collagen ultimate density be respectively 3% (w/w) and 0.1mg/ml solution 1mM and adjust pH value to 7.2 left and right.
By fat stem cell with 10
6the disposable needle tubing that the density of individual/ml is 10ml with the volume of packing into after solid support material solution evenly mixes.
3. connect the non-contact type high voltage static microcapsule generator, high-voltage electrostatic generator adopts SA167-Y (Tianjin) high-voltage electric field producer, output voltage 10kV; Encystation liquid puopulsion unit adopts LongerPump company's T S2-60 type syringe pump, fltting speed 10ml/h, adopt the 10mL syringe, needle point and copper sheet spacing are 30mm, fltting speed is 10ml/h, adopt the syringe needle that internal diameter is 250 μ m, the disposable plastic culture dish that collector is diameter 60mm, solid solution is the 200mmol/L calcium chloride solution.
4. collecting cell microspheres in 5 minutes, with observed and recorded after the physiological saline washed twice, cell microballoon shape rounding is smooth, and median size is about 230 μ m.
With becoming fat nutrient solution (DMEM in high glucose, 1% mycillin stoste, 10% foetal calf serum, 0.5mmol/L isobutyl methylxanthine (IBMX), 1 μ mol/L dexamethasone, 10 μ mol/L Regular Insulin, 200 μ moL/L indomethacins) at 37 ℃, the CO that volume fraction is 0.05
2cultivate the fat stem cell microballoon approximately 10 days in the saturated humidity incubator, obtain the high density adipocyte cluster of sodium alginate/collagen parcel.
The structure of embodiment 2 extracorporeal blood vessel structures
Fat stem cell is become smooth muscle cell to induce: get the fat stem cell of cellar culture between the 4th 0 generations of generation to the, use into the CO that unstriated muscle nutrient solution (containing the M-199 substratum of 5ng/mL TGF-β 1 and 50ng/mLPDGF-BB) is 0.05 in 37 ℃, volume fraction
2in the saturated humidity incubator, cultivate 10 days.Collect 10
8individual smooth muscle cell, standby after centrifugation.
By collagen stoste with becoming the unstriated muscle nutrient solution to be diluted to be slightly larger than after 1mg/ml with the 1mMNaOH neutralization and finally being settled to 1mg/ml.By 10
8individual smooth muscle cell add collagen solution and evenly mix after as coating material.
By 1ml coating material and the centrifuge tube of packing into after 1ml adipocyte microballoon evenly mixes, now the cell microballoon is because density is sunken to more greatly bottom.The centrifugal 5min of the rotating speed of 800r/min, make coating material fully contact with the adipocyte microballoon of bottom.Remove supernatant, add fresh one-tenth unstriated muscle nutrient solution, in centrifuge tube, standing cultivation is 72 hours, make coating material be evenly distributed on microsphere surface and by the self-assembly effect, local microballoon connected into to integral body, form local channel as building microvascular space simultaneously, now can be observed smooth muscle cell and sprawl at microsphere surface.
Fat stem cell is become endotheliocyte to induce: the fat stem cell of getting cellar culture between the 4th 0 generations of generation to the, with becoming the endothelium nutrient solution (containing 2% foetal calf serum, 2% horse serum, 10ng/mL VEGF, 2ng/mL bFGF, 1 * ITS, 0.61g/L nicotinamide, 0.1 μ mol/L dexamethasone, 2g/L BSA, 0.1g/L ornithine, 0.03g/L proline(Pro), 0.73g/L glutamine, 1g/L glucose, the low sugar DMEM substratum of 2g/L semi-lactosi and appropriate trace element (zinc chloride, zinc sulfate and Manganous chloride tetrahydrate etc.)) CO that is 0.05 in 37 ℃, volume fraction
2in the saturated humidity incubator, cultivate 10 days.Collect 10
7individual endotheliocyte, standby after centrifugation.
Add 10 after absorbing the upper strata nutrient solution in centrifuge tube
7individual endotheliocyte, mixing rear pulsation cultivation or common cultivation is that endotheliocyte is combined with coating material and channel surface, and the adipocyte microballoon that continuation pulsation cultivation or common cultivation are compounded with the vascular components cell makes the endotheliocyte that closes on microsphere surface couple together.
Embodiment 3
Adopt the non-contact type high voltage electrostatic power unit to prepare vascularization somatomedin (bFGF and VEGF) sustained-release micro-spheres.
1. sodium alginate is configured to the solution of 1.5% (w/w) with PBS buffered soln.
2. the disposable needle tubing that the concentration of 2ng/ml and 10ng/ml of bFGF and VEGF being take is 10ml with the volume of packing into after encystation solution evenly mixes.
3. connect the non-contact type high voltage static microcapsule generator, operating voltage 7kV, needle point and copper sheet spacing are 20mm, fltting speed is 30ml/h, adopt the syringe needle that internal diameter is 191 μ m, the disposable plastic culture dish that collector is diameter 60mm, solid solution is the 100mmol/L barium chloride solution.
4. collected the somatomedin microballoon in 5 minutes, with observed and recorded after the physiological saline washed twice, the shape rounding of cytokine sustained-release micro-spheres is smooth, and particle diameter is 30 μ m.
5. note keeping low temperature in whole process as far as possible.
Embodiment 4
After the adipocyte microsphere system that is compounded with vascular components is evenly mixed with the volume ratio of 1: 1 with the somatomedin microballoon, pack in the 1ml syringe, the modus operandi of available conventional Autofat granule injection injects in patient body.The final graft formed as shown in Figure 3.
Embodiment 5
The fat stem cell of getting cellar culture adopts the sodium alginate/glutin system as becoming capsule material, with the non-contact type high voltage electrostatic power unit, makes solid-state cell microballoon:
1. the solution of sodium alginate and gelatin solution (solvent PBS buffered soln) are mixed into to the solution that ultimate density is respectively 1.8% (w/w) and 3% (w/w).
By fat stem cell with 10
6the disposable needle tubing that the density of individual/ml is 10ml with the volume of packing into after encystation solution evenly mixes.
3. connect the non-contact type high voltage static microcapsule generator, high-voltage electrostatic generator adopts SA167-Y (Tianjin) high-voltage electric field producer, output voltage 10kV; Encystation liquid puopulsion unit can adopt LongerPump company's T S2-60 type syringe pump, adopt the 5mL syringe, needle point and copper sheet spacing are 10mm, fltting speed is 10ml/h, adopt the syringe needle that internal diameter is 191 μ m, the disposable plastic culture dish that collector is diameter 60mm, solid solution is the 200mmol/L calcium chloride solution.
4. collecting cell microspheres in 5 minutes, with observed and recorded after the physiological saline washed twice, cell microballoon shape rounding is smooth, and particle diameter is 300 μ m.
Adopt into the CO that fat nutrient solution (DMEM in high glucose, 10% foetal calf serum, 0.5mmol/L IBMX, 1 μ mol/L dexamethasone, 10 μ mol/L Regular Insulin, 1% mycillin stoste) is 0.05 in 37 ℃, volume fraction
2in the saturated humidity incubator, the culturing stem cells microballoon is approximately 10 days, obtains the high density adipocyte cluster of sodium alginate/glutin parcel.
Embodiment 6
Fat stem cell is become smooth muscle cell to induce: get the fat stem cell of cellar culture between the 4th 0 generations of generation to the, use into the CO that unstriated muscle nutrient solution (containing the M-199 substratum of 5ng/mL TGF-β 1 and 50ng/mLPDGF-BB) is 0.05 in 37 ℃, volume fraction
2in the saturated humidity incubator, cultivate 10 days.Collect 10
8individual smooth muscle cell, standby after centrifugation.
By solid-state Fibrinogen with becoming the unstriated muscle nutrient solution to be diluted to after 10mg/ml 10
8individual smooth muscle cell add collagen solution and evenly mix after as coating material.
By 1ml coating material and the centrifuge tube of packing into after 1ml adipocyte microballoon evenly mixes, now the cell microballoon is because density is sunken to more greatly bottom.The centrifugal 4min of the rotating speed of 1000r/min, make coating material fully contact with the adipocyte microballoon of bottom.Remove supernatant, add fresh one-tenth unstriated muscle nutrient solution, in centrifuge tube, standing cultivation is 48～72 hours, make coating material be evenly distributed on microsphere surface and by the self-assembly effect, local microballoon connected into to integral body, form local channel as building microvascular space simultaneously, now can be observed smooth muscle cell and sprawl at microsphere surface.
Fat stem cell is become endotheliocyte to induce: the fat stem cell of getting cellar culture between the 4th 0 generations of generation to the, with becoming the endothelium nutrient solution (containing 2% foetal calf serum, 2% horse serum, 10ng/mL VEGF, 2ng/mL bFGF, 1 * ITS, 0.61g/L nicotinamide, 0.1 μ mol/L dexamethasone, 2g/L BSA, 0.1g/L ornithine, 0.03g/L proline(Pro), 0.73g/L glutamine, 1g/L glucose, the low sugar DMEM substratum of 2g/L semi-lactosi and appropriate trace element (zinc chloride, zinc sulfate and Manganous chloride tetrahydrate etc.)) CO that is 0.05 in 37 ℃, volume fraction
2in the saturated humidity incubator, cultivate 10 days.Collect 10
7individual endotheliocyte, standby after centrifugation.
Add 10 after absorbing the upper strata nutrient solution in centrifuge tube
7individual endotheliocyte, mixing rear pulsation cultivation or common cultivation is that endotheliocyte is combined with coating material and channel surface, and the adipocyte microballoon that continuation pulsation cultivation or common cultivation are compounded with the vascular components cell makes the endotheliocyte that closes on microsphere surface couple together.
Embodiment 7 pulsation Process of in vitros
Impulsive motion bioreactor is shown in reference Chinese patent application 200910079726.8 of the present invention.
Employing is originated from the many really peristaltic pump JD-200 of stainless steel accessory service department in Chongyang, Chongzhou City corresponding circulation power is provided, and setting its operating voltage is 12V, and flow velocity is 60ml/min; Direct-current machine is selected the motor ZGB37RH52i that originates from Beijing Aix-en-Provence company, and setting its operating voltage is that 12V, rotating speed are 100r/min; Adopt the syringe of 100ml; To make guide rod and slide block guide rail by oneself, and each parts, as direct-current machine, guide rod, slide block guide rail, syringe are fixed on base plate with homemade support, connecting components.
The nutrient solution cyclic part consists of culture jar, peristaltic pump and cultivation box, and each several part is connected by silicone tube, and nutrient solution is pumped into and cultivates box (built-in artificial fat tissue) from culture jar by peristaltic pump through silicone tube, then through silicone tube, flows back to culture jar; Guide rail slide block, syringe and direct-current machine form the pulsation part, and direct-current machine is connected the pushing syringe reciprocating motion of the pistons with guide rail slide block, after syringe is connected with the outlet end of peristaltic pump, with the cultivation box, are connected, and form thus pulsating flow; Tensimeter is arranged on to be cultivated on box, detects the nutrient solution pressure be placed in the fatty tissue of cultivating in box.
Carrying out before vitro culture, first dismantling pipe connecting, the syringe of impulsive motion bioreactor, utilizing autoclave sterilization.Then connect impulsive motion bioreactor, peristaltic pump and direct-current machine are connected, and at first in culture jar, add a small amount of 75% alcohol, utilize alcohol mobile sterilizing in the pulsation recycle system; Outwell alcohol and then in culture jar, add a certain amount of sterilized PBS solution, utilize this solution cleaning and removing residual alcohol.
Turn off power supply, then in culture jar, add and wait the nutrient solution cultivate to need used, the artificial fatty tissue that good tweezers are clamped embodiment 4 preparations with sterilizing is received on the joint of cultivating box.For artificial organ is connected on joint securely, with the fixing two of artificial tissue of sterilized fine rule.After the impulsive motion bioreactor system connects fully, switch on power, the voltage of adjusting peristaltic pump is 12V, regulates the artificial tissue place 0.1Mpa that is stressed, and then just can the continuous service impulsive motion bioreactor be pulsed and cultivated by artificial tissue.
The pressure that keeps above-mentioned voltage and fatty tissue in culturing process, make in Linear Control pulsation culturing process ripple frequency at 100 times/min.
After direct-current machine operates steadily, band movable slider pushing syringe reciprocating motion of the pistons on guide rail, draw nutrient solution when piston is pulled out from culture jar, while extruding, the nutrient solution sucked is injected to the recycle system that peristaltic pump the forms artificial tissue of cultivating box of flowing through and flow back to culture jar.The amount of the nutrient solution that sucks at every turn, extrudes by the adjusting syringe can be regulated the pressure that cultivator is made the tissue place.Thus, peristaltic pump and direct-current machine persistent movement, impulsive motion bioreactor just provides the nutrient solution stream of a pulsation circulation to realize the pulsation of artificial tissue is cultivated.
Claims (4)
1. a method that builds blood vessel comprises the following steps:
1) autologous stem cells is induced respectively into smooth muscle cell and endotheliocyte, smooth muscle cell evenly mixes with coating material, then add fat tissue cell's microballoon, make it can form at least two-layer in culture space, cultivated, until the coating material that contains smooth muscle cell is evenly distributed on fat tissue cell's microsphere surface, microballoon is connected into to integral body, gap between microballoon forms passage, and described coating material is selected from one or more of collagen, Fibrinogen, matrigel;
2) using the endotheliocyte suspension as nutrient solution, tissue utilization pulsation prepared by step 1) is cultivated or common training method is cultivated, make endotheliocyte be attached to the inwall of described passage, the formation outer wall is the vascular tissue that smooth muscle cell, inwall are endotheliocyte.
2. the method for claim 1, is characterized in that, in step 2) afterwards, also comprise 3) the vascularization somatomedin is wrapped in sodium alginate based aquagel material, make the cytokine sustained-release micro-spheres, then with step 2) homogeneous microstructure that obtains mixes.
3. method as claimed in claim 2, is characterized in that, the described cytokine sustained-release micro-spheres of step 1) described fat tissue cell microballoon and step 3) all adopts the preparation of non-contact type high voltage electrostatic method.
4. method as claimed in claim 2 or claim 3, is characterized in that, the particle diameter of described fat tissue cell microballoon is 100 μ m～500 μ m; The particle diameter of described cytokine sustained-release micro-spheres is 20 μ m～50 μ m.
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| CN109913402B (en) * | 2016-09-14 | 2023-02-21 | 四川蓝光英诺生物科技股份有限公司 | Artificial tissue precursors and methods for making same |
| CN111187749B (en) * | 2018-11-14 | 2021-11-19 | 清华大学 | Artificial structure body for rapidly regenerating vascularized tissue, construction method and application |
| CN111184915B (en) * | 2018-11-14 | 2022-03-18 | 杭州捷诺飞生物科技股份有限公司 | Engineered artificial structure for angiogenesis and construction method and application thereof |
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