CN101366976A - Humanized heterogenous cell epimatrix material and preparation method thereof - Google Patents
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Abstract
The invention relates to a humanized heterogenous extracellular matrix material, which is a dried cellular foam material made by compounding an extracellular matrix and a cell growth factor which are synthesized and excreted by human body cells on a heterogenous extracellular matrix. Cells and natural antigen components are removed from the prepared humanized heterogenous extracellular matrix material, and when the humanized heterogenous extracellular matrix material is applied to the surface of wound, the humanized heterogenous extracellular matrix material can directly take part in the repair of the surface of wound, and the cell growth factor contained in the humanized heterogenous extracellular matrix material can guide the ingrowth of the cells around the wound and creation of blood vessel, and induce the differentiation of stem cells to skin cells, thereby obviously promoting the healing of the surface of wound; besides, the prepared product is a dried product, which greatly prolongs storage life, and not only has part of the characteristics of human body tissues and good mechanical property of the heterogenous extracellular matrix, but also has higher biocompatibility to human body and obviously reduces the immune exclusive reaction; at the same time, the prepared product can cover the surface of wound, fill the defection of soft tissues, promote the growth and proliferation of the cells around the wound, repair the defection of the soft tissue organs, and promote the wound healing.
Description
Technical field
The invention belongs to the technical field of biological materials of organizational project, be specifically related to Humanized heterogenous cell epimatrix material and preparation method thereof.
Background technology
Cell in organ and the tissue, the intrinsic gene order of cell is not only depended in its behavior, also is subjected to the influence of external environment factor to a great extent, comprises the interaction of cell and extracellular matrix.Extracellular matrix not only provides support for the cell growth and protects, and the more important thing is the form generating process of the interaction energy adjusting cell of cell and extracellular matrix, influences cells survival, migration, propagation and functional metabolism.Therefore, the cell epimatrix material that preparation is beneficial to the seed cell adhesion, breeds and breaks up in Tissue Engineering Study is very important and urgent.
(extracellular matrix is by the macromolecular substances of emiocytosis in the extracellular matrix ECM) to extracellular matrix, constitutes complicated grid structure, supports and the conjunctive tissue structure, regulates the generation and the cells physiological activity of tissue.Extracellular matrix be by the synthetic justacrine of body cell to the extracellular, be distributed in the macromole cell surface or the cell, mainly be collagen protein, non-collagen sugar albumen, aminoglycan and Dan Baijutang and elastin laminin.All biosiss such as the shape of extracellular matrix pair cell, structure, function, survival, propagation, differentiation, migration have comprehensive influence, thereby no matter in organ formative process, or in being maintained in body structure and perfect in shape and function all physiological activities of (comprising immunne response and repair in trauma etc.), all has very important important function.
Extracellular matrix can guide, support the host cell growth in vivo, degrades fully gradually, is applicable to the timbering material of organizational project.And contain the various kinds of cell somatomedin in the extracellular matrix, even still contain these somatomedin after cell is handled, can create suitable microenvironment for tissue regeneration at the commitment of wound healing through taking off.This is that other natural material and artificial polymer (as collagen, chitosan and polylactic acid, polyglycolic acid) can not be compared.
The method of obtaining extracellular matrix at present is mainly derived from animal.Yet, the composition of heterogenous cell epimatrix mainly is synthetic by zooblast secretion, has difference with the composition of human body, is difficult to merge with people's bulk phase after therefore implanting, the foreign body reaction of possible excitating organism or immunological rejection in use are restricted it.
Summary of the invention
At the deficiencies in the prior art, the purpose of this invention is to provide a kind of Humanized heterogenous cell epimatrix material and preparation method thereof, make prepared material not only have the feature of heterogenous cell epimatrix, and contain extracellular matrix and multiple somatomedin from the human body cell synthesis secretion, it is damaged and promote wound healing to be used to repair soft tissue organs, has biocompatibility height, nonhazardous, significantly reduces the advantage of immunological rejection.
Humanized heterogenous cell epimatrix material proposed by the invention is to be compounded with the extracellular matrix of human body cell synthesis secretion and the dry porous foam shape material that cell growth factor is constituted on the heterogenous cell epimatrix; Described heterogenous cell epimatrix is with mammalian tissues formed bio-medical material after taking off cell and handling, and can be acellular dermal matrix, takes off the cell fascia, takes off the cell submucous layer of small intestine, takes off the cell submucous layer of bladder, human acellular amniotic membrane, take off the cell cerebral dura mater, takes off cell muscle, takes off the cell tendon, takes off the cell blood vessel, takes off any of cellular neural; Described human body cell is for becoming somatic cell or adult stem cell, comprise Skin Cell, chondrocyte, muscle cell, adipose cell, mesenchymal stem cells MSCs, hematopoietic stem cell, skin progenitor cell, mescenchymal stem cell, muscle stem cell, fat stem cell any or several, its selection is to determine according to the demand in institute repair tissue district.
The preparation method of Humanized heterogenous cell epimatrix material of the present invention comprises that cell obtains, the heterogenous cell epimatrix prepares and the preparation of Humanized heterogenous cell epimatrix material, and concrete steps are as follows:
1), the obtaining of cell: cell is from tissue, selects the type of cell according to the demand in institute repair tissue district, and cell culture technology routinely carries out In vitro culture, and the extensive amplification of cell can adopt the method for cell factory, bioreactor to carry out;
2), the preparation of heterogenous cell epimatrix: the animal skin that obtains or fascia or submucous layer of small intestine or amniotic membrane or muscle or tendon or blood vessel or any neural biomaterial are cut into small pieces, place and contain peracetic acid and alcoholic acid aqueous solution soaking disinfection was not less than 1 hour, with phosphate buffer (PBS) rinsing; If (adopt thicker biomaterial, as dermal layer of the skin, need place-80 ℃ more than freezing half an hour this moment, guarantee that the material internal and external temperature reaches consistent, take out the back and thaw naturally that so multigelation is 2～5 times, make the cell disintegrate of breaking fully, clean) with PBS solution; The NaOH solution soaking that is placed on 0.1～1M is carried out defat, is taken off the cell processing; Embathe neutrality with PBS solution, place the mixed solution that contains DNA enzyme and alpha-galactosidase to soak more than 25 minutes again, remove residual DNA and α-gal native antigen composition, reduce immunogenicity, use the PBS rinsing to pH; For making the easier attaching of repopulating cell, through using any solution soaking of Ox blood serum or collagen protein or poly-D-lysine or arginine-glycine-aspartic acid (rgd peptide) more than 20 minutes behind the dehydrate; Form heterogenous cell epimatrix, sterilization more after drying; Wherein, the percent by volume of described peracetic acid and ethanol final concentration is respectively 0.1～0.5% and 2～10%; The final concentration of described DNA enzyme is 30～50U/ml, and the final concentration of alpha-galactosidase is 10～25U/ml;
3), the preparation of special culture solution, its component by volume percentage ratio includes: commercial minimum essential culture fluid DMEM67.5%, commercial culture fluid F12 22.5%, hyclone 5～15%, insulin 1～50 μ g/ml, basic fibroblast growth factor 1～10ng/ml, hydrocortisone 10～500ng/ml, adenine 0.2～0.25mM, transferrins 1～10 μ g/ml, vitamin C 10～50 μ g/ml.
4), the preparation of Humanized heterogenous cell epimatrix material: the amplification in vitro cultured cells is suspended with cell culture fluid, by 10
4～10
6Individual/cm
2Density drip on heterogenous cell epimatrix surface, at 5%CO
2Leave standstill attaching under 37 ℃ of conditions in the environment, insert and cultivate 2～3 days in the special culture solution; Discard culture fluid, with the heterogenous cell epimatrix not repopulating cell one side upwards, more according to the method described above with 10
4～10
6Individual/cm
2Density repopulating cell (cell of its two sides plantation can be different), leave standstill the back and add special culture solution and cultivated 2～3 days; The heterogenous cell epimatrix that again two sides is compounded with cell places on the cultivation support of cultivating in the vessel, continues to cultivate 6～10 days with special culture solution, changes liquid in per 2 days, and cultivation is finished; Above-mentioned condition of culture is 37 ℃ 5%CO
2Environment;
5), drying: will finish material after the cultivation and be soaked in the frozen solution more than 25 minutes, behind lyophilization, sterilization, obtain being compounded with the extracellular matrix of human body cell synthesis secretion and the dry porous foam shape of the Humanized heterogenous extracellular matrix material of cell growth factor; Described frozen solution is to be added with 0.1～10% glucose, 0.1～5% sucrose, 0.1～5% trehalose, 0.1～0.5% human serum albumin (being weight ratio) in the Hanks balanced salt solution.
The Humanized heterogenous cell epimatrix material of the present invention's preparation, be the compound extracellular matrix components and the various kinds of cell somatomedin of human body cell synthesis secretion on the heterogenous cell epimatrix of the animal origin of removing cell and native antigen composition, make the heterogenous cell epimatrix after the transformation of process human body cell, have the Partial Feature of tissue, not only has heterogenous cell epimatrix favorable mechanical performance, also improved biocompatibility simultaneously, significantly reduced immunological rejection, it is applied to growing into of bootable wound pericyte behind the wound surface, the generation of blood vessel, therefore induced dry-cell can obviously promote the healing of wound surface to the differentiation of Skin Cell.Because freezing dry process not only can make cell inactivation, its storage life prolongs greatly; But the protein in the protective material (protein such as cell growth factor, extracellular matrix) activity is brought into play its normal biological function.Simultaneously can provide three-D space structure for the cell of wound surface, be beneficial to growing into, breeding of cell, the healing that can quicken to organize by the formed dry porous foam shape structure of lyophilization.
The major function of Humanized heterogenous cell epimatrix material of the present invention has: (1) repairs body tissue damaged (malnutrition and infective wound surfaces such as hernia, fistula); (2) make the part plentiful as organizing filler, repair the facial area depressed deformity; (3) be used for the preparation of other organizational project organs as biologic bracket material.
The specific embodiment
Below in conjunction with example technical solution of the present invention is described in further detail.
Example one:
1), the obtaining of fibroblast and epidermis cell: the aseptic application on human skin tissue that obtains, remove fat deposit, be cut into 5mm * 5mm size, after the neutral protein enzymic digestion, separate epidermis and dermal tissue; With 300U/ml collagenase digesting dermal tissue, digestion dispels collecting cell liquid with dermal tissue after finishing, and abandons supernatant after centrifugal to obtain fibroblast, and inoculation is gone into culture bottle and is complemented into the fibrocyte culture fluid, and amplification cultivation is to requirement; Isolating epidermis with after the 0.25% trypsinization liquid digestion, is dispelled collecting cell liquid, abandon supernatant after centrifugal to obtain epidermis cell, inoculation is gone into culture bottle and is supplied the epidermis cell culture fluid, and amplification cultivation is to requirement;
2), the preparation of taking off the cell submucous layer of small intestine: the pig jejunum is clean with distilled water flushing, be cut into the 10cm fragment, place and contained 0.4% (v/v) peracetic acid and the alcoholic acid aqueous solution soaking disinfection of 8% (v/v) 1 hour; With longitudinally cutting open after phosphate buffer (PBS) rinsing, mechanical curettage mucous membrane of small intestine, flesh layer and serous coat after the PBS rinsing, obtain tela submucosa; Insert again in the NaOH solution of 0.4M and to take off cell under 4 ± 2 ℃ of conditions and handled 1 hour, with the PBS rinsing to pH value neutrality; It is inserted in the mixed solution of alpha-galactosidase of the DNA enzyme that contains 40U/ml and 20U/ml, 37 ℃ of environment are handled half an hour down, remove residual DNA and α-gal native antigen composition, use the PBS rinsing, behind dehydrate, use hyclone to soak 30 minutes, after lyophilization, ethylene oxide sterilizing is sterilized again;
3), the preparation of special culture solution, each component by volume percentage ratio is: commercial minimum essential culture fluid DMEM67.5%, commercial culture fluid F1222.5%, hyclone 10%, insulin 10 μ g/ml, basic fibroblast growth factor 2ng/ml, hydrocortisone 15ng/ml, adenine 0.2mM, transferrins 8 μ g/ml, vitamin C 40 μ g/ml.
4), the preparation of Humanized heterogenous cell epimatrix material: fibroblast and epidermis cell digestion, centrifugal with In vitro culture suspend with cell culture fluid respectively, by 10
5Individual/cm
2Density fibroblast is dripped taking off cell submucous layer of small intestine surface, in 5%CO
2Left standstill 30 minutes under 37 ℃ of conditions in the environment, add special culture solution and cultivated 2 days, discard culture fluid, will take off the cell submucous layer of small intestine not repopulating cell one side upwards, more according to the method described above with 10
5Individual/cm
2Density plantation epidermis cell, leave standstill the back and add special culture solution and cultivated 3 days, the heterogenous cell epimatrix that again two sides is compounded with cell places on the cultivation support of cultivating in the vessel, continues to cultivate 8 days with special culture solution, changes liquid, and promptly cultivates and finish in per 2 days; Above-mentioned condition of culture is 37 ℃ 5%CO
2Environment;
5), drying: will finish Humanized heterogenous cell epimatrix material after the cultivation and be soaked in the frozen solution 30 minutes; Frozen solution is to be added with 1% glucose, 2% sucrose, 1% trehalose, 0.1% human serum albumin (being weight ratio) in the Hanks balanced salt solution; After vacuum lyophilization, the ethylene oxide sterilizing sterilization obtains freeze dried Humanized heterogenous cell epimatrix material.
The Humanized heterogenous cell epimatrix material of this examples preparation includes the multiple Humanized cell somatomedin and the extracellular matrix components of human fibroblasts and epidermis cell synthesis secretion, comprise bFGF, EGF, TGF-β 1, IL-8, collagen protein etc., can be directly used in that to repair soft tissue organs damaged and promote wound healing, can suppress cicatrization and wound surface is reinvented.
Example two:
1), fat stem cell obtains: the aseptic human fat tissue that obtains, be cut into 1mm
3Size after the digestion of 400U/ml collagenase liquid, dispels collecting cell liquid with fatty tissue, abandons supernatant after centrifugal to obtain fat stem cell; Add the outstanding cell of cell culture fluid after-blow, culture bottle is gone in inoculation, and amplification cultivation is to requirement;
2), take off the preparation of cell Corii Sus domestica substrate: it is laminar that the Corii Sus domestica skin that will remove fat deposit is cut into 5cm * 10cm, clean with distilled water flushing, places to contain 0.2% peracetic acid and 3% alcoholic acid aqueous solution soaking disinfection 1.5 hours; After the phosphate buffer rinsing, place-80 ℃ freezing 40 minutes, make the Corii Sus domestica internal and external temperature reach consistent, take out the back and thaw naturally, multigelation like this 3 times makes the cell disintegrate of breaking fully; With the NaOH solution soaking that is placed on 0.8M after the washed with de-ionized water 1 hour; Reuse PBS liquid embathes to pH value neutrality, is placed in the mixed solution of alpha-galactosidase of the DNA enzyme of 50U/ml and 25U/ml 1 hour, removes residual DNA and α-gal native antigen composition, cleans with PBS; Behind dehydrate, soaked 50 minutes at 0.1% Poly-L-Lysine Solution; Promptly become through lyophilization again and take off cell Corii Sus domestica substrate, cobalt 60 sterilizations.
3), the preparation of special culture solution, each component by volume percentage ratio is: commercial minimum essential culture fluid DMEM67.5%, commercial culture fluid F12 22.5%, hyclone 10%, insulin 40 μ g/ml, basic fibroblast growth factor 5ng/ml, hydrocortisone 100ng/ml, adenine 0.22mM, transferrins 6 μ g/ml, vitamin C 30 μ g/ml.
4), the preparation of Humanized heterogenous cell epimatrix material: with the fat stem cell digestion of In vitro culture, centrifugal after, suspend again with cell culture fluid, by 10
6Individual/cm
2Density drip after taking off cell Corii Sus domestica stromal surface, in 5%CO
2Left standstill 30 minutes under 37 ℃ of conditions in the environment, add special culture solution and cultivated 2 days, discard culture fluid, will take off cell Corii Sus domestica substrate not repopulating cell one side upwards, more as stated above with 10
5Individual/cm
2Density plantation fat stem cell, leave standstill the back and add special culture solution and cultivated 3 days, again its taking-up is placed on the cultivation support of cultivating in the vessel, continue to cultivate 8 days with special culture solution, changed liquid in per 2 days between culture period, promptly finish cultivation; Above-mentioned condition of culture is 37 ℃, 5%CO
2Environment.
5), drying: will finish material after the cultivation and be soaked in the frozen solution 50 minutes; Frozen solution is to add 5% glucose, 0.5% sucrose, 4% trehalose, 0.2% human serum albumin (being weight ratio) in the Hanks balanced salt solution; Through lyophilization, after the ethylene oxide sterilizing sterilization, obtain freeze dried Humanized heterogenous cell epimatrix material.
The somatomedin that the Humanized heterogenous cell epimatrix material of this examples preparation contains the fat stem cell synthesis secretion can influence fibroblast and quicken wound healing, is used for the treatment of the healing of soft tissue defects.
Example three:
1), fibroblastic obtaining: with example one;
2), take off the preparation of cell submucous layer of small intestine: with example one;
3), the preparation of special culture solution, each component by volume percentage ratio is: commercial minimum essential culture fluid DMEM 67.5%, commercial culture fluid F12 22.5%, hyclone 10%, insulin 20 μ g/ml, basic fibroblast growth factor 10ng/ml, hydrocortisone 200ng/ml, adenine 0.25mM, transferrins 10 μ g/ml, vitamin C 50 μ g/ml.
4), the preparation of Humanized heterogenous cell epimatrix material: fibroblast is suspended with cell culture fluid, by 10
6Individual/cm
2Density drip taking off cell submucous layer of small intestine surface, in 5%CO
2Left standstill 30 minutes under 37 ℃ of conditions in the environment, add special culture solution and cultivated 3 days, discard culture fluid, will take off the cell submucous layer of small intestine not repopulating cell one side upwards, more according to the method described above with 10
6Individual/cm
2Density plantation fibroblast, leave standstill the back and add special culture solution cultivation 2 days, the cell submucous layer of small intestine that takes off that again two sides is compounded with cell places on the cultivation support of cultivating in the vessel, continue to cultivate 10 days with special culture solution, changed liquid, and finished the cultivation of Humanized heterogenous cell epimatrix material in per 2 days; Above-mentioned condition of culture is 37 ℃, 5%CO
2Environment;
5), drying: will finish Humanized heterogenous cell epimatrix material after the cultivation and be soaked in the frozen solution 30 minutes; Frozen solution is to be added with 0.5% glucose, 2% sucrose, 1% trehalose, 0.5% human serum albumin (being weight ratio) in the Hanks balanced salt solution; Through vacuum lyophilization, behind cobalt 60 sterilizations, obtain freeze dried Humanized heterogenous cell epimatrix material.
The lyophilizing Humanized heterogenous cell epimatrix material of this examples preparation can be preserved 12 months after cell inactivation processing, lyophilization, be convenient to clinical use, it contains the multiple Humanized cell somatomedin and the extracellular matrix components of abundant human fibroblasts synthesis secretion, as bFGF, TGF-β 1, IL-8, collagen protein etc., can participate in granulation tissue and cicatrization and the later stage wound surface is reinvented, it is damaged and promote wound healing to can be used for repairing soft tissue organs.
Claims (5)
1. a Humanized heterogenous cell epimatrix material is characterized in that, is to be compounded with the extracellular matrix of human body cell synthesis secretion and the dry porous foam shape material that cell growth factor is constituted on the heterogenous cell epimatrix; Described heterogenous cell epimatrix is mammalian tissues formed bio-medical material after taking off the cell processing; Described human body cell is for becoming somatic cell or adult stem cell.
2. the method for preparing the described Humanized heterogenous cell epimatrix material of claim 1 includes the step that cell obtains, and it is characterized in that concrete steps are as follows:
1), the obtaining of cell: select the type of cell according to the demand in institute repair tissue district, cell culture processes routinely carries out In vitro culture and extensive amplification;
2), the preparation of heterogenous cell epimatrix: the animal skin that obtains or fascia or submucous layer of small intestine or amniotic membrane or muscle or tendon or blood vessel or any neural biomaterial are cut into small pieces, place and contain peracetic acid and alcoholic acid aqueous solution soaking disinfection was not less than 1 hour; Use the phosphate buffer rinsing; After placing the NaOH solution soaking to handle, embathe neutrality, place the mixed solution that contains DNA enzyme and alpha-galactosidase to soak more than 25 minutes again, use the phosphate buffer rinsing to pH with phosphate buffer; Through using any solution soaking of Ox blood serum or collagen protein or poly-D-lysine or arginine-glycine-aspartic acid more than 20 minutes behind the dehydrate; Form heterogenous cell epimatrix, sterilization more after drying;
3), the preparation of special culture solution, its component by volume percentage ratio includes: commercial minimum essential culture fluid DMEM 67.5%, commercial culture fluid F12 22.5%, hyclone 5～15%, insulin 1～50 μ g/ml, basic fibroblast growth factor 1～10ng/ml, hydrocortisone 10～500
Ng/ml, adenine 0.2～0.25mM, transferrins 1～10 μ g/ml, vitamin C 10～50 μ g/ml;
4), the preparation of Humanized heterogenous cell epimatrix material: the amplification in vitro cultured cells is suspended with cell culture fluid, by 10
4～10
6Individual/cm
2Density drip on heterogenous cell epimatrix surface, at 5% CO
2After leaving standstill attaching under 37 ℃ of conditions in the environment, insert and cultivate 2～3 days in the special culture solution; Discard culture fluid, with the heterogenous cell epimatrix not repopulating cell one side upwards, more according to the method described above with 10
4～10
6Individual/cm
2The density repopulating cell, leave standstill the back and add special culture solution and cultivated 2～3 days; The heterogenous cell epimatrix that again two sides is compounded with cell places on the cultivation support of cultivating in the vessel, continues to cultivate 6～10 days with special culture solution, changes liquid in per 2 days, and cultivation is finished; Above-mentioned condition of culture is 37
05% CO of C
2Environment;
5), drying: will finish material after the cultivation and be soaked in the frozen solution more than 25 minutes; behind lyophilization, sterilization, obtain being compounded with the extracellular matrix of human body cell synthesis secretion and the dry porous foam shape of the Humanized heterogenous extracellular matrix material of cell growth factor.
3. preparation method according to claim 2 is characterized in that, in step 2) described in the final concentration volume ratio of peracetic acid be 0.1～0.5%, alcoholic acid final concentration volume ratio is 2～10%; The final concentration of described DNA enzyme is 30～50U/ml, and the final concentration of alpha-galactosidase is 10～25U/ml.
4. preparation method according to claim 2, it is characterized in that, in step 2) described in soaking disinfection clean after, before the NaOH solution soaking, material is placed-80 ℃ more than freezing half an hour, take out the back and thaws naturally, so multigelation is 2～5 times, with after the phosphate buffer cleaning again in the NaOH solution soaking.
5. preparation method according to claim 2; it is characterized in that, be to be added with 0.1～10% glucose, 0.1～5% sucrose, 0.1～5% trehalose, 0.1～0.5% human serum albumin in the Hanks balanced salt solution by weight at the frozen solution described in the step 5).
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