CN1304566C - Complex organ-like precursor with culture device and structure thereof and culturing method - Google Patents
Complex organ-like precursor with culture device and structure thereof and culturing method Download PDFInfo
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Abstract
The present invention relates to a complex organ-like precursor with a culturing device, a constructing method of the complex organ-like precursor with a culturing device, and a culturing method. The present invention adopts computer software for designing complex organ-like precursor molds of the three-dimensional structure and forming paths according to the people's organ structure and functional characteristics; then seed cells and substrate material mixing objects are stacked into a blank of the three-dimensional class organ by using a quickly forming or other cell assembling techniques; auxiliary culturing apparatus, such as inner and outer air bags or bioreactors are used for carrying out exercise and inducting culture for the blank of the class organs to make the class organs convert to functional organs. The present invention imitates the structure and functions of the complicated organs to realize the construction of the complex organ-like precursors on the three-dimensional sizes. The present invention promotes the cells in the complicated organs arranged according to one direction to enhance the mechanical strength of the organ precursors to make the organ precursors have a plurality of special functions by a controllable three-dimensional stress field and the exercise or the training of the bioreactors. Simultaneously, the present invention fulfills cell proliferation growth and metastatic requirements, and the complex organ-like precursors have the trend for converting to natural organs.
Description
Technical field
The present invention relates to a kind of design and construction process thereof of complex organ precursor, belong to the bioengineered tissue technical field.
Background technology
Annual in the world patient's number of suffering from tissue defect or organ failure exceedes ten million, and only U.S. every year is with this type of patient of surgical operation therapy about 8,000,000.Yet live body donor tissue organ is limited, and the existing mechanical device does not possess all functions of complicated tissue organ, can not prevent that patient's the state of an illness from further worsening.In view of the above, arise at the historic moment with organizational project (Tissue Engineering) technology that to improve this type of illness treatment level be aim.Organizational project is a new and high technology subject that is produced by multidisciplinary intersection such as biology, medical science, materialogy, engineering science.Its implication is to use the philosophy and technique of life science and engineering science, on the weave construction under the mammiferous normal and pathology two states of correct understanding and emic basis, research, develop the function that is used to repair, safeguard, promote behind various tissues of human body or the organ damage and biological substitution thing [the Merem RM.Med ﹠amp of form; BiolEng ﹠amp; Comput.1992; 30:8-12].Over past ten years, scientists utilization tissue engineering technique, utilize a small amount of normal cell of human body rudimentary organ to carry out external breeding, obtain patient's organ required, that have identical function, there is not rejection again, got gratifying achievement, many biotech companies that set up are recently just preparing to drop into huge fund and are realizing commercialization.In the U.S., formed 4,000,000,000 dollars the industry that is worth, and with annual 25% speed increase.Surpass 1,000 ten thousand dollars of [Miller M, Biotech Bioeng, 1996 as the Pro Osteon coral bone grafting material output value; 50:4347-4348].But existing tissue engineering technique faces many difficulties and restriction, and the obtained success of organizational project applied research all is comparatively simply to organize as bone, cartilage in those structures and physiological function.Traditional support technology of preparing is the passage of size, structure, spatial distribution and the perforation of control punch exactly, and nutrition supply and blood vessel are grown into and all be very restricted.The general preparation earlier of tradition Method of Tissue Engineering structure stand, in carrying out cell cultivation process since most oxygen of upper strata cell consumption and nutrition limited these components and spread to bottom, thereby limited cell to the migration of support deep layer etc.This support of preparation earlier, the method for culturing cell again, effort again consuming time, cell modification, aging just probably in the process of in support, moving, the requirement that does not reach timely treatment clinical patient.Traditional tissue engineering technique of while can not satisfy accurately location and the fixed point placement in the space of different cells, makes up the demand of the function gradient structure of complicated tissue organ.
Summary of the invention
The purpose of this invention is to provide a kind of complex organ-like precursor and structure and cultural method that has culture apparatus, be intended on the basis of previous work, utilize microcomputer modelling, groups of cells packing technique, directly cell and extracellular matrix biomimetic material are assembled into three-dimensional structure, make complex organ-like precursor under the effect of 3-D stree field by culture apparatus, iuntercellular is connected and induced growth, form functional organization, make this type of organ precursor to the transition of functional type organ, thereby reach the purpose of reparative regeneration, be with a wide range of applications.
Technical scheme of the present invention is as follows:
The invention provides a kind of complex organ-like precursor that has culture apparatus, it is characterized in that: described complex organ-like precursor links to each other with an auxiliary culture apparatus, described complex organ-like precursor is the organoid cavity of the three-dimensional structure that surrounded by smooth surface, its chamber wall is made up of cell and substrate material, is provided with a plurality of liquid access ways on the wall of chamber; Described auxiliary culture apparatus comprises and is arranged on outer air bag of the intravital air bag in chamber and cavity and the gas compressing apparatus that links to each other with inside and outside air bag, is filled with cell culture fluid between air bag and the interior air bag outside.
Another kind provided by the invention has the complex organ-like precursor of culture apparatus, it is characterized in that: described complex organ-like precursor links to each other with an auxiliary culture apparatus, described complex organ-like precursor is the organoid cavity of the three-dimensional structure that surrounded by smooth surface, its chamber wall is made up of cell and substrate material, on the wall of chamber, be provided with liquid-inlet and liquid exit, in cavity, be provided with the TRS that cell and substrate material are formed; Described auxiliary culture apparatus comprises the bio-reactor of pulsation or rotation, and the liquid entrance of described organoid precursor links to each other with the fluid circulation system of described bio-reactor.
The complex organ-like precursor culture apparatus of another kind of three-dimensional structure provided by the invention, its feature also is: be provided with the liquid fluid channel on the TRS of described cell and substrate material composition.
A kind of structure and cultural method that has the complex organ-like precursor of culture apparatus provided by the invention is characterized in that this method comprises the steps:
1) sets up the complex organ-like precursor model of three-dimensional structure
According to the structure and the functional characteristics of human organ, utilize corresponding calculated machine Autocad to design the complex organ-like precursor model and the forming path of three-dimensional structure;
2) preparation of seed cell and substrate material miscellany
Preparation concentration is 1～40% aseptic substrate material solution; Buy or extract seed cell, with after the suspension centrifugation of different types of seed cell of gained respectively with the solution uniform mixing that contains 1～40% substrate material, make the mixture of cell and substrate material; Described substrate material is gelatin, collagen, gelatin/collagen, collagen/chitosan, gelatin/chitosan or gelatin/sodium alginate, the mass ratio 0.1～100: 1 of collagen and chitosan, gelatin and chitosan or gelatin and sodium alginate in the mixing solutions of wherein said collagen/chitosan, gelatin/chitosan or gelatin/sodium alginate, described seed cell accounts for 1～90% of described substrate material mixing solutions; The solution of described substrate material is the sodium-chlor of 0.09M of the aqueous solution or pH=6～8 or sodium-chlor and the 3-hydroxymethyl aminomethane hydrochloric acid soln of 0.09M; Also can add 1～10% serum, 0.0001～0.1% cell growth factor and extracellular matrix mixture (Matrigel), heparin, chondroitin sulfate, the hyaluronic acid of gene or 0.1～100% in the described substrate material;
3) the three-dimensional controlled stack shaping of seed cell and substrate material miscellany
Forming path according to model in the step 1), utilize fast shaping technology, adopt the digital composite spraying technique of single shower nozzle or many shower nozzles, adopt discrete/build up process, to step 2) in the mixture of seed cell and substrate material disperse/stack shaping, formation has 1) in the blank of organoid precursor of the defined three-dimensional structure of model, this three-dimensional organoid precursor blank is handled through physical condensation, Chemical Crosslinking Methods, obtain three-dimensional complex organ-like precursor;
4) with air bag is set inside and outside the complex organ-like precursor, between inside and outside air bag, inject cell culture fluid; Inside and outside air bag is connected with gas compressing apparatus; Make inside and outside airbag inflation or contraction by gas compressing apparatus, make complex organ-like precursor be subjected to the effect of three dimensional stress, cell culture fluid is flowed between the inside and outside of cavity by the fluid passage on the cavity, make between each confluent monolayer cells under the effect of controlled three dimensional stress, to set up closely to connect and induce its growth, form functional organ.
Another kind provided by the invention has the structure and the cultural method of the complex organ-like precursor of culture apparatus, it is characterized in that this method comprises the steps:
1) sets up the complex organ-like precursor model of three-dimensional structure
According to the structure and the functional characteristics of human organ, utilize corresponding calculated machine Autocad to design the complex organ-like precursor model and the forming path of three-dimensional structure;
2) preparation of seed cell and substrate material miscellany
Preparation concentration is 1～40% aseptic substrate material solution; Buy or extract seed cell, with after the suspension centrifugation of different types of seed cell of gained respectively with the solution uniform mixing that contains 1～40% substrate material, make the mixture of cell and substrate material; Described substrate material is gelatin, collagen, gelatin/collagen, collagen/chitosan, gelatin/chitosan or gelatin/sodium alginate, the mass ratio 0.1～100: 1 of collagen and chitosan, gelatin and chitosan or gelatin and sodium alginate in the mixing solutions of wherein said collagen/chitosan, gelatin/chitosan or gelatin/sodium alginate, described seed cell accounts for 1～90% of described substrate material mixing solutions; The solution of described substrate material is the sodium-chlor of 0.09M of the aqueous solution or pH=6～8 or sodium-chlor and the 3-hydroxymethyl aminomethane hydrochloric acid soln of 0.09M; Also can add 1～10% serum, 0.0001～0.1% cell growth factor and extracellular matrix mixture (Matrigel), heparin, chondroitin sulfate, the hyaluronic acid of gene or 0.1～100% in the described substrate material;
3) the three-dimensional controlled stack shaping of seed cell and substrate material miscellany
Forming path according to model in the step 1), utilize fast shaping technology, adopt the digital composite spraying technique of single shower nozzle or many shower nozzles, adopt discrete/build up process, to step 2) in the mixture of seed cell and substrate material disperse/stack shaping, formation has 1) in the blank of organoid precursor of the defined three-dimensional structure of model, this three-dimensional organoid precursor blank is handled through physical condensation, Chemical Crosslinking Methods, obtain three-dimensional complex organ-like precursor;
4) complex organ-like precursor is placed the annular seal space of bio-reactor of pulsation or rotation, the liquid entrance of organoid precursor is linked to each other with the fluid circulation system of described bio-reactor; Make complex organ precursor be subjected to the effect of three dimensional stress by pulsation or rotation, make closely contact under the effect of controlled three dimensional stress between each confluent monolayer cells, iuntercellular connects and induced growth, forms functional organization; Satisfy cell proliferation growth and metabolic needs simultaneously.
The present invention compared with prior art, have the following advantages and the high-lighting effect: the present invention utilizes the installation of microcomputer modelling, groups of cells can realize that different cells and substrate material accurately locate at spatial, having overcome the present inducing culture cell in three-dimensional rack that exists of organizational project needs the time long, cell is skewness in support, and cell is difficult to penetrate into the medium shortcoming of deep structure.The present invention imitates the 26S Proteasome Structure and Function of complex organ, realized the structure of complex organ-like precursor on the three dimension scale, and training or cultivation by controlled 3-D stree field and bio-reactor, impel in the complex organ cell to arrange by a certain direction, improve the physical strength of organ precursor, made it to have some special functions.Satisfied cell proliferation growth and metabolic needs simultaneously, the transformation of organoid precursor to natural organ's direction arranged.As simulating the chamber structure of heart, realized the blood-pumping function of heart, thereby reached the purpose of repairing heart damage cardiac muscular tissue or whole atrium organ, improving its function.
Description of drawings
Fig. 1 is the structural representation of embodiment that has the class heart precursor of air bag culture apparatus.
Fig. 2 is the structural representation of class liver precursor.
Fig. 3 is the structural representation that has the class liver precursor of bio-reactor.
Embodiment
Fig. 1 a kind of structural representation that has the class heart precursor of air bag culture apparatus provided by the invention.Described class heart precursor links to each other with an auxiliary culture apparatus, described complex organ-like precursor is the class heart cavity 1 of the three-dimensional structure with rotational symmetry and opening that surrounded by smooth surface, its chamber wall 2 is made up of cell and substrate material, is provided with a plurality of liquid access ways 3 on the wall of chamber; Described auxiliary culture apparatus comprise be arranged on the chamber intravital in the outer outer air bag 5 of air bag 4 and cavity and the gas compressing apparatus 6 that links to each other with inside and outside air bag, be filled with cell culture fluid 7 between air bag and the interior air bag outside.In air bag, add a certain amount of gas, pilot-gas flowing in inside and outside air bag, thereby the contraction or expansion of the inside and outside air bag of control.When outer airbag inflation, when interior air bag shrinks, the organoid precursor is subjected to the effect of three-dimensional stress, shrinks in the chamber, and nutritive medium is being flowed in the export-oriented chamber by the chamber by fluid passage 3 simultaneously.When outer air bag shrinks, during interior airbag inflation, the organoid precursor is subjected to the effect of three-dimensional tensile stress, outside the chamber, expand, simultaneously nutritive medium by fluid passage by outside the chamber, flowing in the chamber.Through training after a while, make this type of organ precursor under the effect of 3-D stree field, make closely contact under the effect of controlled three-dimensional normal stress between each confluent monolayer cells on the one hand, impel iuntercellular to set up mutual connection, form functional organization; Promote the circulation of nutritive substance in this type of organ precursor on the other hand, satisfied cell proliferation growth and metabolic needs.
Fig. 2 is the structural representation of class liver precursor.Fig. 3 is the structural representation that has the class liver precursor of bio-reactor.Described class liver precursor links to each other with an auxiliary culture apparatus, described class liver precursor is the cavity 1 of the class liver precursor of the three-dimensional structure that surrounded by smooth surface, its chamber wall is made up of cell and substrate material, on the wall of chamber, be provided with liquid-inlet 8 and liquid exit 9, in cavity, be provided with the TRS 10 that cell and substrate material are formed; Described auxiliary culture apparatus comprises the bio-reactor 11 and a liquor pump 12 that links to each other with this bio-reactor of pulsation or rotation, and described liquid entrance links to each other with the fluid circulation system of described bio-reactor.By the fluctuating force of bio-reactor or the effect of revolving force, make cell in this type of liver precursor by the regular arrangement of a certain direction and set up connection, form functional liver organization.
Concrete implementation step of the present invention is as follows:
1) has the foundation of the complex organ precursor model of three-dimensional structure
According to the structure and the functional characteristics of human organ (as heart, liver, kidney etc.), utilize corresponding calculated machine Autocad (as SolidWorks) to design a kind of complex organ precursor model with axisymmetric sphere, ellipse or asymmetric oblateness, the special three-dimensional structure of leaf shape.The feature of this model is: it is that certain thickness cell and substrate composed thin-walled are arranged that the three-dimensional structure that a kind of symmetry or asymmetric smooth surface surround, this structure have cavity, on the wall of chamber liquid entrance is arranged.The pipe network structure that can contain perforation in the thin-walled to guarantee the circulation of nutrient solution (vitro culture) or blood (body is implanted into), can be with porous on the wall of chamber.
2) preparation of seed cell and substrate material miscellany
Buy or extract seed cell, selected biomaterial is dissolved in the solution, preparation concentration is 1～40% aseptic substrate material solution.With after the suspension centrifugation of different types of seed cell of gained respectively with the solution uniform mixing that contains 1～40% substrate material, make the mixture of cell and substrate material; Described substrate material is gelatin, collagen, gelatin/collagen, collagen/chitosan, gelatin/chitosan or gelatin/sodium alginate, the mass ratio 0.1～100: 1 of gelatin and chitosan or gelatin and sodium alginate in the mixing solutions of wherein said collagen/chitosan, gelatin/chitosan or gelatin/sodium alginate, described seed cell accounts for 1～90% of described substrate material mixing solutions; The solution of described substrate material is the sodium-chlor of 0.09M of the aqueous solution or pH=6～8 or sodium-chlor and the 3-hydroxymethyl aminomethane hydrochloric acid soln of 0.09M; Also can add 1～10% serum, 0.0001～0.1% cell growth factor and the extracellular matrix of gene or 0.1～100% in the described substrate material and mix (Matrigel), heparin, chondroitin sulfate, hyaluronic acid;
3) the three-dimensional controlled stack shaping of seed cell and substrate material miscellany
According to 1) middle model definition forming path, utilize fast shaping technology, adopt the digital composite spraying technique of single or many shower nozzles, adopt discrete/build up process, to 2) in the blend of seed cell and substrate material disperse/stack shaping, form have 1) blank of the organoid precursor of the middle defined three-dimensional axially symmetric structure of model.This three-dimensional organoid precursor is handled through methods such as physical condensation, chemically crosslinkeds, made it in considerable time, to keep Stability Analysis of Structures.
4) cell in the organoid precursor is along the training and the external evoked cultivation of controlled 3-D stree field
With 3) in the organoid precursor that makes insert in the specific device, make wherein cell be subjected to the effect of controlled 3-D stree field, iuntercellular is set up and is connected and induced growth, finally trends towards forming specific function.Concrete grammar is as follows:
A) with one and 3) in make the interior air bag sterilization that the intracoelomic cavity size conforms to before the organoid after, put into inner chamber by the upper end open of this type of organ precursor; Then total system is put into outer air bag.Between inside and outside air bag, inject the organoid precursor and cultivate necessary nutritive medium (as Fig. 1).In air bag, add a certain amount of gas, make airbag inflation to a constant volume.By computer control system, pilot-gas flowing in inside and outside air bag, thereby the contraction of the inside and outside air bag of control.When outer airbag inflation, interior air bag shrank, the organoid precursor was subjected to the effect of three-dimensional stress, in the chamber, shrink, simultaneously nutritive medium by porous and blood vessel access by flowing in the export-oriented chamber, chamber.When outer air bag shrinks, during interior airbag inflation, the organoid precursor is subjected to the effect of three-dimensional tensile stress, outside the chamber, expand, simultaneously nutritive medium by porous and blood vessel access by outside the chamber, flowing in the chamber.Through training after a while, make this type of organ precursor under the effect of 3-D stree field, make closely contact under the effect of controlled three-dimensional normal stress between each confluent monolayer cells on the one hand, impel iuntercellular to set up mutual connection, formative tissue; Promote the circulation of nutritive substance in this type of organ precursor on the other hand, satisfied cell proliferation growth and metabolic needs.Like this, under the multiple action of stress field, iuntercellular connects and induced growth, makes this type of organ precursor trend towards forming specific function.
B), adopt bio-reactor that it is carried out inducing culture (Fig. 3,37 ℃, 5%CO for many blood vessels such as liver, kidney complex organ
2).Wherein bio-reactor can be flowing-type, pulsating or rotary.Also whole bio-reactor can be placed on CO
2In the incubator it is carried out inducing culture.
The several embodiment that enumerate below are with the further the present invention of understanding
Embodiment 1: the preparation of class cardiac muscle chamber precursor
1) foundation of class cardiac muscle chamber precursor 3 d structure model
According to the structure and the functional characteristics of heart, utilize corresponding calculated machine Autocad (as SolidWorks) to design a kind of class heart cardiac muscle chamber precursor model (Fig. 1) with axisymmetric special three-dimensional structure.
2) preparation of seed cell and substrate material miscellany
Extract endotheliocyte, myocardial cell and fibroblastic mixture, type i collagen is dissolved in 1% the acetum by weight 0.4%, heart endothelial cell, myocyte and fibroblastic mixture and collagen solution, extracellular matrix Matrigel (0.1%) aqueous solution were evenly mixed by 10: 1: 1, make cell and substrate material miscellany, regulate miscellany pH to 7 with sodium hydroxide solution (0.1N).
3) the three-dimensional controlled accumulation moulding and the external evoked cultivation of cell and substrate material miscellany
According to 1) middle model definition forming path, utilize fast shaping technology, adopt the digital composite spraying technique of single shower nozzle, adopt discrete/build up process, to 2) in the blend of cell and substrate material disperse/stack shaping, formation has 1) in the blank of class cardiac muscle chamber precursor of the defined three-dimensional axially symmetric structure of model, this structure has the cavity of hollow.Cavity is by certain thickness cell and substrate composed thin-walled, and upper end open can be with porous on the wall of chamber.This three-dimensional class cardiac muscle chamber precursor was solidified 45 minutes through physics in 37 ℃, 5% CO2 incubator, adds the fresh nutrient solution continuation of being rich in serum 7 days.
4) class cardiac muscle chamber is along the training of controlled 3-D stree field
With one and 3) in make the air bag sterilization that the intracoelomic cavity size conforms to before the class heart after, put into inner chamber by the upper end open of this type of myocardium chamber precursor.Outside the chamber of this type of myocardium chamber precursor, also wrap up the sterilization air bag of a corresponding size.Then total system is put into cavity volumes such as a sealing.Wait in the cavity volume in this sealing, inject class cardiac muscle chamber precursor and cultivate necessary nutritive medium (as Fig. 1).In air bag, add a certain amount of gas, make airbag inflation arrive the formulation volume.By computer control system, pilot-gas flowing in inside and outside air bag, thereby the contraction of the inside and outside air bag of control.When outer airbag inflation, interior air bag shrank, class cardiac muscle chamber precursor was subjected to the effect of three-dimensional stress, in the chamber, shrink, simultaneously nutritive medium by porous and blood vessel access by outside the chamber, flowing in the chamber.When outer air bag shrinks, during interior airbag inflation, class cardiac muscle chamber precursor is subjected to the effect of three-dimensional tensile stress, outside the chamber, expand, simultaneously nutritive medium by porous and blood vessel access by flowing in the export-oriented chamber, chamber.Through training after a while, make this type of myocardium chamber precursor under the effect of 3-D stree field, make closely contact under the effect of controlled three-dimensional normal stress between each confluent monolayer cells on the one hand, impel iuntercellular to set up mutual connection, formative tissue; Promote the circulation of nutritive substance in this type of organ precursor on the other hand, satisfied cell proliferation growth and metabolic needs.Like this, under the multiple action of stress field, iuntercellular connects and induced growth, and has the autonomous contractility of three-dimensional, makes this type of myocardium chamber precursor trend towards forming the specific function of heart.
Embodiment 2: the preparation of atrium precursor
1) foundation of class cardiac muscle chamber precursor 3 d structure model
According to the structure and the functional characteristics of heart, utilize corresponding calculated machine Autocad (as SolidWorks) to design a kind of class atrium precursor model (Fig. 1) with axisymmetric special three-dimensional structure.
2) preparation of seed cell and substrate material miscellany
Buy and to have built embryonic stem cell, the endotheliocyte that is, (100: 1w/w) solution is made embryonic stem cell, endotheliocyte and substrate material miscellany with 30% gelatin/chitosan respectively with embryonic stem cell, endotheliocyte.The myocardial cell's somatomedin or the endothelial cell growth factor (ECGF) of adding 0.1% in embryonic stem cell, endotheliocyte and substrate material miscellany mix respectively.
3) the three-dimensional controlled accumulation moulding and the external evoked cultivation of embryonic stem cell, endotheliocyte and substrate material miscellany
According to 1) middle model definition forming path, utilize fast shaping technology, adopt the digital composite spraying technique of two shower nozzles, adopt discrete/build up process, to 2) in the blend of embryonic stem cell, endotheliocyte and substrate material, somatomedin disperse/stack shaping, form have 1) blank of the class atrium precursor of the middle defined three-dimensional axially symmetric structure of model.This three-dimensional organoid precursor was handled 10 minutes through 3% sodium polyphosphate, used 0.2% glutaraldehyde water solution more crosslinked 2 minutes, make it in considerable time, to keep Stability Analysis of Structures.
4) class cardiac muscle chamber is along the training of controlled 3-D stree field
With one and 3) in make the air bag sterilization that the intracoelomic cavity size conforms to before the class atrium after, put into inner chamber by the upper end open of this type of heart precursor.Outside the chamber of this type of heart precursor, also wrap up the sterilization air bag of a corresponding size.Then total system is put into a closed cavities.In this closed cavities, inject class heart precursor and cultivate necessary nutritive medium (as Fig. 1).In air bag, add a certain amount of gas, make airbag inflation arrive the formulation volume.By computer control system, pilot-gas flowing in inside and outside air bag, thereby the contraction of the inside and outside air bag of control.When outer airbag inflation, interior air bag shrank, interior heart precursor was subjected to the effect of three-dimensional stress, in the chamber, shrink, simultaneously nutritive medium by porous and blood vessel access by flowing in the export-oriented chamber, chamber.When outer air bag shrinks, during interior airbag inflation, class atrium precursor is subjected to the effect of three-dimensional tensile stress, outside the chamber, expand, simultaneously nutritive medium by porous and blood vessel access by outside the chamber, flowing in the chamber.Through training after a while, make this type of atrium precursor under the effect of 3-D stree field, make closely contact under the effect of controlled three-dimensional normal stress between each confluent monolayer cells on the one hand, impel iuntercellular to set up mutual connection, formative tissue; Promote the circulation of nutritive substance in this type of organ precursor on the other hand, satisfied cell proliferation growth and metabolic needs.Like this, under the multiple action of stress field, iuntercellular connects and induced growth, and has the autonomous contractility of three-dimensional, makes this type of atrium precursor trend towards forming the specific function in atrium.This type of atrium precursor can directly be attached to disease and decrease on the liver, plays the purpose of reparative regeneration.
Embodiment 3: the preparation of class liver precursor
1) foundation of class liver precursor model:, design a kind of liver precursor area of computer aided CAD (Computer-aided design) model (Fig. 2) of simplification with pipe network structure according to the structure and the functional characteristics of liver.
2) preparation of seed cell and substrate material miscellany
Adopt patient's liver separation and Culture to obtain liver cell (comprising liver stem cells), stellate cell, bile duct epithelial cell or its cell mixing, endotheliocyte, (1: 0.09M NaCI solution 100w/w) is compound respectively, preparation cell and substrate material miscellany with endotheliocyte, several compound liver cell and gelatin/chitosan.
3) the three-dimensional controlled accumulation moulding and the external evoked cultivation of cell and substrate material miscellany
According to 1) middle model definition forming path, utilize fast shaping technology, adopt the digital composite spraying technique of single shower nozzle, adopt discrete/build up process, to 2) in the blend of compound cells and substrate material disperse/stack shaping, form have 1) blank of the class liver precursor of the middle defined three-dimensional axially symmetric structure of model.This three-dimensional class liver precursor through 0.1% glutaraldehyde cross-linking, is made it to keep Stability Analysis of Structures in considerable time.
4) vitro culture of class liver precursor model
With 3) in make class liver precursor and be placed on (Fig. 3) in the impulsive motion bioreactor, allow flow through pipeline in the precursor of nutrient solution.Through the training in a week, make this type of liver precursor under the effect of liquid flow power, cell is by the regular arrangement of a certain direction and set up connection, forms hepatic tissue.
Embodiment 4: the preparation of artificial liver precursor
The bone marrow stem cell that separates patient, vitro culture, amplification.With the different sorts cell suspension centrifugal after respectively or the sodium-chlor Tutofusin tris hydrochloric acid soln that mixes with the 0.09M of 40% gelatin mix, prepare the multitube road 3-D solid structure that bone marrow stem cell/substrate material is assembled into by the groups of cells installation.Glutaraldehyde solution with 1% reacts 30 seconds, normal saline flushing.This structure is placed in the rotating biological reactor and cultivated for two weeks, can directly be attached to disease and decrease on the liver, plays the purpose of reparative regeneration.
Embodiment 5: the preparation of artificial liver precursor
Buy and to have built the embryonic stem cell that is, liver cell gene etc., external evoked, cultivate, amplification.(100: aqueous solution 1w/w) is even with 20% gelatin/sodium alginate respectively after centrifugal with the different sorts cell suspension, the pHGF of adding 0.1%, by groups of cells installation preparation stem cell, liver cell gene, cell growth factor and the substrate composed multitube of gelatin/sodium alginate road 3-D solid structure, elder generation crosslinked 20 minutes with 10% calcium chloride, use for 0.1% crosslinked 10 seconds of glutaraldehyde physiological saline again, be put in the impulsive motion bioreactor and cultivate.
Embodiment 6: the preparation of breast precursor
The separating out fat cell, vitro culture, amplification.(100: 1w/w) 0.09M NaCI solution, adipocyte somatomedin mix with 1% gelatin/chondroitin sulfate respectively after centrifugal with the adipocyte suspension, prepare umbrella room shape breast precursor with Rapid Prototyping technique, by 0.25% glutaraldehyde water solution crosslinked 2 minutes, cultivated for two weeks with the rotating biological reactor, this complex body can directly be planted in the injury of breast place.
Embodiment 7: the preparation of breast precursor
The separating out fat stem cell, vitro culture, amplification.(1: 1w/w) solution mixes with 10% collagen/gelatin with the centrifugal back of fat stem cell suspension, the three-dimensional structure for preparing multitube road cell and matrix by the shape of breast by Rapid Prototyping technique, with 0.02% glutaraldehyde water solution crosslinked 3 minutes, soaked 30 minutes with containing 0.1% heparin solution again, cultivated for two weeks with impulsive motion bioreactor, this complex body can directly be planted in the injury of breast place.
Embodiment 8: the preparation of kidney precursor
Separate the nephrocyte gene, vitro culture, amplification.(100: 1w/w) aqueous solution, cell growth factor mix with 1% gelatin/sodium alginate with nephrocyte gene (DNA), the kidney shape precursor for preparing diameter 0.5 μ m with the groups of cells packing technique, with 0.3% glutaraldehyde water solution crosslinked 1 minute, cultivated for two weeks with impulsive motion bioreactor.This complex body can directly be planted in the injury of the kidney place.
Embodiment 9: the preparation of kidney precursor
Separate nephrocyte, vitro culture, amplification.The nephrocyte suspension is mixed with weight ratio 30% gelatin 0.09M NaCI solution (pH:6) respectively after centrifugal, prepare multitube road kidney shape three-dimensional structure by Rapid Prototyping technique, 0.3% glutaraldehyde water solution crosslinked 2 seconds, with containing 0.1% hyaluronic aqueous solution soaking 30 minutes, after the impulsive motion bioreactor cultivation, directly plant in body.
Embodiment 10: the preparation of pancreas precursor
Separate islet cells, vitro culture, amplification.The islet cells suspension is mixed with weight ratio 20% aqueous gelatin solution, Regular Insulin respectively after centrifugal, crosslinked with the webmaster shape of Rapid Prototyping technique preparation by 0.3 glutaraldehyde water solution.After cultivating a week with impulsive motion bioreactor, this complex body can directly be planted in positions such as pancreas, livers.
Embodiment 11: the preparation of pancreas precursor
Separate the islet cells gene, vitro culture, amplification.(1: 2w/w) 0.09M NaCI pH value of solution: 7.4, Regular Insulin mixes the three-dimensional structure that is prepared multitube road cell and gelatin/sodium alginate matrix by Rapid Prototyping technique with weight ratio 20% gelatin/sodium alginate with the islet cells gene, crosslinked 10 minutes of 5% calcium lactate solution, used 0.25% glutaraldehyde normal saline solution more crosslinked 1 minute, and planted in body after cultivating with the rotating biological reactor.
Embodiment 12: the preparation of pancreas precursor
Separate the pancreas islet gene, vitro culture, amplification.With pancreas islet gene and weight ratio 1% gelatin/chitosan (100: 1w/w) 0.09M NaCI three (methylol) aminomethane hydrochloric acid soln (pH:8.0), mix, the three-dimensional structure for preparing multitube road cell and matrix by Rapid Prototyping technique, crosslinked 20 seconds of 0.1% glutaraldehyde normal saline solution, after cultivating a week with impulsive motion bioreactor, can directly plant in body or vitro culture for some time implants again.
Embodiment 13: the preparation of lung precursor
Separate pulmonary epithelial cells, pneumonocyte, vitro culture, amplification.(1: 0.09M NaCI solution (pH ≈ 7.4) 1w/w), cell growth factor mix with weight ratio 1% gelatin/sodium alginate respectively after centrifugal with the isolated cell suspension, with two shower nozzle groups of cells installation preparation dendroid lung precursors, by glutaraldehyde cross-linking, impulsive motion bioreactor is cultivated.
Embodiment 14: the preparation of lung precursor
Separate pulmonary epithelial cells, pneumonocyte.(1: 2w/w) solution (pH:6), cell growth factor mix with weight ratio 10% gelatin/chitosan respectively after centrifugal with pulmonary epithelial cells, pneumonocyte suspension, the three-dimensional structure for preparing flat garden shape multitube road cell and matrix by Rapid Prototyping technique, used 0.1% glutaraldehyde normal saline solution after 5% Tri sodium Phosphate is crosslinked more crosslinked 20 seconds, heparin solution with 1% was soaked 30 minutes, and impulsive motion bioreactor cultivated for 2 weeks.
Embodiment 15: the preparation of spleen precursor
Separating Morr. cell, endotheliocyte, vitro culture, amplification.Splenocyte, endotheliocyte suspension are mixed with 20% gelatin/chitosan (pH:7.4) solution respectively after centrifugal, class spleen precursor with two shower nozzle groups of cells installation preparation cells and substrate material, crosslinked by 0.2% glutaraldehyde normal saline solution, the outer circulation of rotating biological reactor body is cultivated, and grafts in injury of spleen place.
Claims (2)
1. complex organ-like precursor that has culture apparatus, it is characterized in that: described complex organ-like precursor links to each other with an auxiliary culture apparatus, described complex organ-like precursor is the organoid cavity of the three-dimensional structure that surrounded by smooth surface, its chamber wall is made up of cell and substrate material, is provided with a plurality of liquid access ways on the wall of chamber; Described auxiliary culture apparatus comprises and is arranged on outer air bag of the intravital air bag in chamber and cavity and the gas compressing apparatus that links to each other with inside and outside air bag, is filled with cell culture fluid between air bag and the interior air bag outside.
2. a construction process that has the complex organ-like precursor of culture apparatus as claimed in claim 1 is characterized in that this method comprises the steps:
1) set up the complex organ-like precursor model of three-dimensional structure:
According to the structure and the functional characteristics of human organ, utilize corresponding calculated machine Autocad to design the complex organ-like precursor model and the forming path of three-dimensional structure;
2) preparation of seed cell and substrate material miscellany:
Preparation concentration is 1～40% aseptic substrate material solution; Buy or extract seed cell, with after the suspension centrifugation of different types of seed cell of gained respectively with the solution uniform mixing that contains 1～40% substrate material, make the mixture of cell and substrate material; Described substrate material is gelatin, collagen, gelatin/collagen, collagen/chitosan, gelatin/chitosan or gelatin/sodium alginate, the mass ratio 0.1～100: 1 of collagen and chitosan, gelatin and chitosan or gelatin and sodium alginate in the mixing solutions of wherein said collagen/chitosan, gelatin/chitosan or gelatin/sodium alginate, described seed cell accounts for 1～90% of described substrate material mixing solutions; The solution of described substrate material is the sodium-chlor of 0.09M of the aqueous solution or pH=6～8 or sodium-chlor and the 3-hydroxymethyl aminomethane hydrochloric acid soln of 0.09M;
3) the three-dimensional controlled stack shaping of seed cell and substrate material miscellany:
Forming path according to model in the step 1), utilize fast shaping technology, adopt the digital composite spraying technique of single shower nozzle or many shower nozzles, adopt discrete/build up process, to step 2) in the mixture of seed cell and substrate material disperse/stack shaping, formation has 1) in the blank of organoid precursor of the defined three-dimensional structure of model, this three-dimensional organoid precursor blank is handled through physical condensation, Chemical Crosslinking Methods, obtain three-dimensional complex organ-like precursor;
4) with air bag is set inside and outside the complex organ-like precursor, between inside and outside air bag, inject cell culture fluid; Inside and outside air bag is connected with gas compressing apparatus; Make inside and outside airbag inflation or contraction by gas compressing apparatus, make complex organ precursor be subjected to the effect of three dimensional stress, cell culture fluid is flowed between the inside and outside of cavity by the fluid passage on the cavity, make closely contact under the effect of controlled three dimensional stress between each confluent monolayer cells, iuntercellular connects and induced growth, forms functional organization; Satisfy cell proliferation growth and metabolic needs simultaneously.
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