CN105664258A - Method for preparing smooth muscle cell carrier based on small intestine submucosa acellular matrix - Google Patents
Method for preparing smooth muscle cell carrier based on small intestine submucosa acellular matrix Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3629—Intestinal tissue, e.g. small intestinal submucosa
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3679—Hollow organs, e.g. bladder, esophagus, urether, uterus, intestine
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- A—HUMAN NECESSITIES
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3882—Hollow organs, e.g. bladder, esophagus, urether, uterus
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0661—Smooth muscle cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
Abstract
The invention discloses a method for preparing a smooth muscle cell carrier based on a small intestine submucosa acellular matrix. Vagina smooth muscle cells are successfully cultured in vitro, and the cultured vagina smooth muscle cells are seen to be in a long fusiform and gathered on a culture dish to form a typical peak and valley structure when invertedly placed under a microscope. The submucosa acellular matrix is white in appearance and translucent and has certain tenacity. Hematoxylin-eosin dying is conducted, and no cell component exists. After a vagina smooth muscle cell-intestine submucosa specimen section is dyed by hematoxylin-eosin, the number of visible cells under a light microscope is gradually increased, and the visible cells grow from the surface to the deep layer. After the vagina smooth muscle cell-intestine submucosa specimen section is dyed by an anti-rabbit smooth muscle alpha-actin monoclonal antibody in an immunohistochemical mode, positive cells of anti-rabbit alpha-Actin can be seen. The result preliminarily proves that the pig intestine submucosa acellular matrix can be taken as the smooth muscle cell carrier.
Description
Technical field
The present invention relates to a kind of method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix, belong to medical skin tissue engineering technique field.
Background technology
Bio-derived material has the structure similar to extracellular matrix and good biocompatibility, is a kind of tissue engineering bracket material having superiority very much. In numerous bio-derived materials, submucous layer of small intestine (SIS) has the advantages such as reduced immunogenicity, antibiotic property and promotion tissue growth, so being considered as a kind of ideal tissue engineering bracket material.
Containing abundant proteoglycan, collagen protein and fibronectin splicing variants in SIS, sticking, breed and breaking up between cell and tissue can be promoted. Proteoglycan is everlasting the commitment of cell adhesion, and attachment and stretching, extension stage such as cell work; Collagen protein α 1 chain by type i collagen, fibronectin splicing variants is by RGD sequence and cell-membrane receptor specific bond, and activation signal pathway promotes cell adhesion.
The multiple somatomedin that SIS comprises, as VEGF (VEGF), basic fibroblast growth factor (b-FGF) etc. have important facilitation in the reparation of tissue and reconstruct. SIS has the anatomical structure of uniqueness: the Heparin sulfate proteoglycan between collagen fiber is concentrated mainly on blood vessel; In Dispersed precipitate; VEGF is then mainly distributed on around blood vessel. This structure plays a significant role in tissue regeneration.
The defects such as the domestic and international main support material polyglycolic acid existence degraded for vagina tissue engineering research at present is too fast. Natural de-cell scaffold material especially small intestinal submucosa is increasingly becoming the emphasis of Tissue Engineering Study.
Vagina is the important genitals of women, due to the part or all of disappearance that the reasons such as congenital diseases or the damage day after tomorrow, tumor cause, it is necessary to carry out repairing and reproducing. Apply various autologous or allosome tissue and carry out vagina when repairing, not only can cause new damage, and postoperative it being likely to occur the phenomenons such as vaginal reconstruction contracture, excess secretion and abnormal flavour, the vagina additionally reproduced all has very big-difference with normal vagina tissue in histology, morphology and function aspects.If a small amount of cell of patient self can be utilized, gone out the vagina tissue of access expansion by tissue engineering technique " reproducing ", then cover vaginal cavity chamber, be then undoubtedly a kind of novel, desirable vaginoplasty.
2003, vaginal epithelial cell and smooth muscle cell are planted and are successfully constructed organizational project vagina tissue on the polyglycolic acid support being coated with 50:50 different amount Molecularly Imprinted Polymer (left-handed and dextrorotation interior friendship fat and glycolic hand over lipopolymer) by DeFilippo etc., plant vaginal epithelial cell thereon and vagina smooth muscle growth and proliferation of cell is good. But polyglycolic acid material immunogenicity, antimicrobial originality, can promote the characteristics such as tissue regeneration compared with natural scaffold materials also have a certain distance. Small intestinal submucosa (smallintestinalsubmucosa, SIS) substrate is as natural extracellular matrix class bio-derived material, it is cut its Submucosa from pig small intestine solution to obtain the biomaterial rich in collagen after treatment, there is non-immunogenicity, antimicrobial originality, cell regeneration can be promoted, cells survival and the three dimensions sprawled are provided, it is beneficial to the characteristics such as cell metabolism, experiment and clinical position are relatively used for the reparation of the tissue defects such as blood vessel, bladder, nerve and stomach wall, it is shown that good histocompatibility. The pig SIS such as Rotariu repairs rabbit up to 2.5cm Urethral defect, it was observed that SIS can promote the regeneration of urothelial, histological examination see in substrate newborn urethral tissue layer almost with normal indifference. Urothelial Cell and bladder smooth muscle cells are planted on SIS by Gabouev etc., and SABC shows and defines urothelium and bladder smooth muscle cells double-decker, is successfully made for engineered bladder. The present invention prepares the SIS cellular matrix of acellular composition, cultivation is expanded vagina smooth muscle cell seeding on SIS, external compound criteria observation of cell sticks on the material, breeds, intrusion situation, to inquire into the pig SIS feasibility as tissue engineering vagina smooth muscle cell carrier, lay the foundation for developing acellular matrix tissue engineering vagina.
Summary of the invention
Purpose: in order to overcome the deficiencies in the prior art, the present invention provides a kind of method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix.
Technical scheme: for solving above-mentioned technical problem, the technical solution used in the present invention is:
A kind of method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix, comprises the following steps:
1)Prepared by SIS:
Physical method processes: small intestinal takes from weight > the family pig of 200kg; By small intestinal freezed storage in transportation, in 4h, complete cleaning process; Placenta percreta and the muscle layer of small intestinal is removed, therebetween persistently with 40 DEG C of warm water washings with the handle of a knife being wrapped with gauze;
Chemical method processes: cuts the small intestinal processed through physical method along the longitudinal axis, is cut into every section of 15cm length, prepares by Abraham method; Each step of chemical method processing procedure all at room temperature carries out, and the ratio that material amasss with solution is held at 1:100;
2)The separation and Culture of vagina smooth muscle cell: draw materials, separate: choose 2.0 ~ 2.5kg doe, after ketamine 25mg/kg intramuscular injection anesthesia, fixing limbs, fully expose cloacal aperture; Hypogastric region and external genital iodophor disinfection, vagina rinses with aseptic metronidazole liquid; Above pubic symphysis, do about 5cm otch along hunter's line, cut skin, rectus abdominis m. and peritoneum successively, mention vagina, it is seen that the vagina tissue being connected with double uterus, after completely cutting vagina tissue, sewing-up cut, successively closes abdomen;Repeatedly rinse, with physiological saline solution, dual anti-liquid, the vagina tissue taken off, specimen is put in the dual anti-liquid of the PBS+ being made ready beforehand for afterwards, be sent to laboratory; Vagina tissue block is soaked in 5min in 0.05% hibitane by superclean bench, again by the moistening piece of tissue of DMEM, carefully separating removal mucosal epithelium layer and serous coat connective tissue layer with blade and ophthalmic operating set, then tiling to prune by the smooth muscle tissue of separator well forms 1mm3Piece of tissue, and make piece of tissue neat in edge, smooth;
3)Original cuiture: piece of tissue+enzyme digestion carries out original cuiture; The piece of tissue pruned is digested 0.5 ~ 1.0h with 0.1% collagenase IV in 37 DEG C of incubators, digestion is stopped when becoming hair to piece of tissue edge, then the piece of tissue digested is inoculated on culture dish, interval is about 5mm, quiescent culture 3.0 ~ 4.0h in 37 DEG C of constant incubators, after tissue block adherent is firm, add containing the DMEM culture fluid that volume fraction is 10% hyclone again, 37 DEG C of constant temperature quiescent culture 3.0 ~ 4.0d, change liquid after cell is swum out of, and change weekly liquid twice or thrice to maintain Growth of Cells;
4)Vagina smooth muscle cell is planted on SIS:Go down to posterity when Growth of Cells reaches 80% fusion, discard old culture medium, mixture slaking liquid is added to culture bottle, hatch 3-7min for 37 DEG C, observe that intercellular substance increases, cell is rounded, add the hyclone that volume fraction is 0.8% when being partially suspended in Digestive system in culture dish and terminate digestion, by centrifugal for cell suspension 5min, centrifugal collecting cell, culture medium settling flux, with 5.0 × 106/cm2Be inoculated on SIS substrate inner face, 37 DEG C, volume fraction be 5%CO2Quiescent culture in incubator; Culture medium is still containing the DMEM culture fluid that volume fraction is 10% hyclone, changes culture medium once every 2d.
Preferably, chemical method processes and specifically refers to: (1) is at the middle immersion under 37 DEG C of conditions containing 0.5mmol/L ethylenediaminetetraacetic acid (EDTA) and 0.4% tryptic solution, stirring 5.0 ~ 6.0h; (2) continue, with 0.2% glutaraldehyde, tissue is carried out crosslinking to protect; Rinse well with deionized water, be placed in the solution containing 40 μm of ol/LDNA enzymes and 1mmol/L sodium chloride soak, stirring pig small intestine 6 ~ 8h; (3) with soaking in sodium chloride (1mmol/L) phosphate buffer (PBS) after deionized water rinsing, stirring 16h; (4) with soaking 2h after deionized water rinsing in PBS solution (pH value is 7.0 ~ 7.4); (5) again with deionized water rinsing substrate 2h; (6) sterilization: SIS is soaked in 20% alcoholic solution containing 0.1% peracetic acid 8h, with containing 0.05% Hydrazoic acid,sodium salt PBS solution cleaning 2h, can short-term preservation in 4 DEG C of PBS solution.
Preferably, the described method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix, it is characterised in that: chemical method processing procedure, except sterilization, carries out under all immersions in magnetic stirring apparatus, stirring in all of processing procedure.
Preferably, the described method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix, it is characterised in that: described dual anti-liquid is the mixed solution of penicillin and streptomycin.
Preferably, the described method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix, it is characterised in that: described mixture slaking liquid is 0.25% pancreatin: 0.02%EDTA=1:1.
Preferably, the described method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix, it is characterised in that: described centrifugal rotational speed is 1000r/min, centrifugal radius 13cm.
Beneficial effect: the method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix provided by the invention, 1. external vagina smooth muscle cell is successfully turned out, under inverted microscope, seeing that the vagina smooth muscle cell of cultivation presents long shuttle shape, cell assembles typical " peak and valley " the sample configuration of formation on culture dish. 2. Submucosa acellular matrix white in appearance, translucent, has certain toughness. Hematoxylin-eosin staining has no cell component to be existed. 3., after vagina smooth muscle cell-intestinal mucosa lower floor specimen section hematoxylin-eosin staining, under light microscopic, visible cell composition increases gradually, is grown to deep layer position by table is shallow. 4. after vagina smooth muscle cell-intestinal mucosa lower floor specimen section adopts anti-rabbit Smooth muscle α-actin monoclonal antibody immunity histochemical staining, the positive cell of all visible anti-rabbit α-Actin. Result preliminary proof intestinal mucosa underlying substrate can as a kind of smooth muscle cell carrier.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described.
Embodiment 1:
SIS preparation and histological examination:
Physical method processes: small intestinal takes from weight > the family pig of 200kg. By small intestinal freezed storage in transportation, in 4h, complete cleaning process. Placenta percreta and the muscle layer of small intestinal is removed, therebetween persistently with 40 DEG C of warm water washings with the handle of a knife being wrapped with gauze.
Chemical method processes: cuts the small intestinal processed through physical method along the longitudinal axis, is cut into every section of 15cm length, prepares by methods such as Abraham. Each step of chemical method processing procedure all at room temperature carries out, and the ratio that material amasss with solution is held at 1:100.
Method particularly includes: (1) at the middle immersion under 37 DEG C of conditions containing 0.5mmol/L ethylenediaminetetraacetic acid (EDTA) and 0.4% tryptic solution, stirring 5.0 ~ 6.0h. (2) continue, with 0.2% glutaraldehyde, tissue is carried out crosslinking to protect; Rinse well with deionized water, be placed in the solution containing 40 μm of ol/LDNA enzymes and 1mmol/L sodium chloride soak, stirring pig small intestine 6 ~ 8h. (3) with soaking in sodium chloride (1mmol/L) phosphate buffer (PBS) after deionized water rinsing, stirring 16h. (4) with soaking 2h after deionized water rinsing in PBS solution (pH value is 7.0 ~ 7.4). (5) again with deionized water rinsing substrate 2h. (6) sterilization: SIS is soaked in 20% alcoholic solution containing 0.1% peracetic acid 8h, with containing 0.05% Hydrazoic acid,sodium salt PBS solution cleaning 2h, can short-term preservation in 4 DEG C of PBS solution. Above except sterilization process, all of processing procedure carries out under all immersions in magnetic stirring apparatus, stirring.
SIS histological examination: use optical microscope and hematoxylin-eosin staining to observe the histological structure of SIS after physics and chemical method process.
The separation and Culture of vagina smooth muscle cell: draw materials, separate: choose 2.0 ~ 2.5kg new zealand female rabbit, after ketamine 25mg/kg intramuscular injection anesthesia, fixing limbs, fully expose cloacal aperture. Hypogastric region and external genital iodophor disinfection, vagina rinses with aseptic metronidazole liquid. Above pubic symphysis, do about 5cm otch along hunter's line, cut skin, rectus abdominis m. and peritoneum successively, mention vagina, it is seen that the vagina tissue being connected with double uterus, after completely cutting vagina tissue, sewing-up cut, successively closes abdomen. Repeatedly rinse, with physiological saline solution, dual anti-liquid, the vagina tissue taken off, afterwards specimen is put in the dual anti-liquid of the PBS+ being made ready beforehand for (penicillin+streptomycin), be sent to laboratory.Vagina tissue block is soaked in 5min in 0.05% hibitane by superclean bench, again by the moistening piece of tissue of DMEM, carefully separating removal mucosal epithelium layer and serous coat connective tissue layer with blade and ophthalmic operating set, then tiling to prune by the smooth muscle tissue of separator well forms 1mm3Fritter, and make piece of tissue neat in edge as far as possible, smooth. Piece of tissue+enzyme digestion carries out original cuiture.
Original cuiture: the piece of tissue pruned is digested 0.5 ~ 1.0h with 0.1% collagenase IV in 37 DEG C of incubators, digestion is stopped when becoming hair to piece of tissue edge, then the piece of tissue digested is inoculated on culture dish, interval is about 5mm, quiescent culture 3.0 ~ 4.0h in 37 DEG C of constant incubators, after tissue block adherent is firm, add containing the DMEM culture fluid that volume fraction is 10% hyclone again, 37 DEG C of constant temperature quiescent culture 3.0 ~ 4.0d, change liquid after cell is swum out of, and change weekly liquid twice or thrice to maintain Growth of Cells. Inverted microscope observation of cell form and growing state.
Rabbit vagina smooth muscle cell is planted on SIS: go down to posterity when Growth of Cells reaches 80% fusion, discard old culture medium, mixture slaking liquid (0.25% pancreatin: 0.02%EDTA=1:1) is added to culture bottle, hatch about 5min for 37 DEG C, observe that intercellular substance increases, cell is rounded, add the hyclone that volume fraction is 0.8% when being partially suspended in Digestive system in culture dish and terminate digestion, by centrifugal for cell suspension (1000r/min, centrifugal radius 13cm) 5min, centrifugal collecting cell, culture medium settling flux, with 5.0 × 106/cm2Be inoculated on SIS substrate inner face, 37 DEG C, volume fraction be 5%CO2Quiescent culture in incubator. Culture medium is still containing the DMEM culture fluid that volume fraction is 10% hyclone, changes culture medium once every 2d.
The rabbit vagina smooth muscle cell growth status being inoculated in pig SIS is observed under inverted microscope: take the SIS-cell conjugate after inoculation 1,2,3,4 week respectively, rinse 3 times with 0.01% phosphate buffer, put into 2.5% glutaraldehyde solution fixing, send microscopy together with fixative in the lump.
Pathological observation: take respectively 1,2,3,4 week SIS-cell conjugate specimen conventional-volume mark be 10% formalin fix, paraffin embedding, after section, row hematoxylin-eosin staining.
Anti-rabbit a-actin antibodies carries out immunohistochemical staining detection a-Actin (SABC method): take respectively 1,2,3,4 week SIS-cell conjugate specimen conventional-volume mark be 10% formalin fix, paraffin embedding, after section, row immunohistochemical staining.
MAIN OUTCOME MEASURES: 1. SIS histological examination. 2. vagina smooth muscle cell in-vitro growth feature. 3. the rabbit vagina smooth muscle cell growth status on pig SIS. 4. Immunohistochemical detection smooth muscle a-expression of actin.
SIS histological examinationSIS after physics and chemical method process, substantially sees white translucent. Under light microscopic, by many queueing disciplines, the webbed collagen fiber of compact siro spinning technology form the host material of preparation; Hematoxylin-eosin staining takes on a red color, and inside does not find cell debris.
Vagina smooth muscle cell in-vitro growth featureUnder inverted microscope, primary cell is adherent, growth gradually, and cell size does not wait, various shapes, has fusiformis, strip, banding triangle etc., and nucleus is oval or circle, has 1 ~ 3 not of uniform size, dark kernel in core. Amplification along with cell, Growth of Cells scope constantly expands, covering at the bottom of culture bottle to 16 ~ 20d cell, Growth of Cells is intensive, in shuttle-type or long shuttle-type, cytoplasmic process rises interlaced, fusion together, subregion cell multiple-layer overlapped, subregion cell monolayer, height rises and falls, typical " peak-to-valley " shape growth structure feature in Smooth Muscle Cell.
Rabbit vagina smooth muscle cell growth status on pig SIS
Under inverted microscope, after being inoculated into SIS upper 1 week, a few cell is had to start to attach on SIS, form similar round, elongated, elapse in time, attach cell number and increase, part cytochrome oxidase isozymes becomes fusiformis. When SIS-cell conjugate is cultivated in culture dish, long fusiform cell from complex around eruption in culture dish epontic phenomenon be can be observed, even if the SIS-cell conjugate of the 4th week is also such.
Hematoxylin-eosin staining is observed
Dyeing showed cell plantation after the 1st week, smooth muscle cell is predominantly located at the table superficial part position of SIS acellular matrix. Passage cell quantity showed increased in time, it is seen that cell invades to the middle part of SIS acellular matrix.
Immunohistochemical detection Smooth muscle α-actin is expressed
Anti-rabbit α-Actin is the monoclonal antibody of anti-α-actin, and anti alpha-Actin dyeing identifies that cultured cells is brown color for the positive, and this is specific to smooth muscle cell. It is seeded in the vagina smooth muscle cell anti-rabbit a-Actin immunohistochemical staining on intestinal mucosa lower floor support material positive.
Adopting the concentration of 0.4% and the digestion time of 5.0 ~ 6.0h, EDTA Main Function is in that to combine with divalent ion such as Ca2+, Mg2+ etc., promotes to separate in cell self-organizing, and to collagen stroma not damaged. The EDTA digestion of different organization need variable concentrations, tests the EDTA digestion that have employed 0.5mmol/L for pig jejunum construction features, had both reached de-cell fully, and had protected again the effect of collagen stroma. Finally employing DNA enzymatic, it can be hydrolyzed by catalytic nucleic acid, eliminates the intramatrical nucleic acid compositions of small intestinal.
Sterilizing after SIS preparation: the biomaterial for all animal origins, a potential problem is that they can carry the pathogen of danger. Although many virulence factors can be destroyed by extreme pH or humidity, but still have some virulence factors such as gastral cavity virus that chemically or physically degraded is had extreme resistance. For avoiding the infection of zoonosis, typically by peracetic acid disinfectant, 0.1% peracetic acid (being 20% dehydrated alcohol containing volume fraction) is soaked 8h and can be reached complete disinfective action. Research confirms to process pig small intestine and SIS with this disinfectant solution, and measure pod membrane virus, without pod membrane virus, including pig parvoviral, porcine respiratory enterovirus, murine leukemia retroviral and PRV (Pseudorabies virus), prove that pod membrane virus is prone to remove, 5min can inactivate, after processing 30min, all viruses are all inactivated. After sterilized, sterilizing place, it is desirable to the microorganism total amount in SIS is dropped to each below specimen 100cfu, and endotoxin drops to each below specimen 20EU.
Storage after SIS preparation:After the SIS sterilizing of preparation can short term stored 4 DEG C of cold preservation in PBS solution, if desired for long term storage, first want Programmed cryopreservation to-80 DEG C, lyophilizing, dried three layers vacuum packaging, then with 2.5 × 10-5Gy gamma-rays illumination-based disinfection, be stored in 4 DEG C standby. But this experiment row short term storage, therefore gamma-rays illumination-based disinfection useless. Experiment employing is vacuum-packed is primarily intended to deoxygenation, rotten to desirably prevent SIS, its principle is also fairly simple, because SIS Biodeterioration is mainly caused by the activity of microorganism, the existence of most of microbe needs oxygen, and this principle is used in vacuum packaging exactly, oxygen in packaging bag and in SIS is taken out, makes microorganism lose " environment of existence ". Experiment proves: when oxygen purity≤1% in packaging bag, microbial growth and reproduction speed just sharply decline, and during oxygen purity≤0.5%, most of microbe is subjected to suppress and stop breeding.Vacuum packaging can not suppress the rotten and variable color that the breeding of anaerobe and enzyme reaction cause completely, therefore also needs to be combined with other householder methods, as adopted cold preservation, quick-freezing, gamma-rays illumination-based disinfection etc. Deaeration in condenser is except suppressing microbial growth and breeding, and another critical function is to prevent SIS from aoxidizing.
The external Combined culture of smooth muscle cell-SIS
Smooth muscle cells inoculation opportunity: external structure organizational project vagina, it is crucial and core procedure that seed cell is inoculated on three-dimensional stent material, cell inoculation, for three-dimensional distribution in support of initiator cell amount, cell in timbering material and cell proliferation and differentiation subsequently, migrates and the aspect such as organization formation has material impact. Therefore the inoculation of organizational project cell requires higher, first has to ensure higher rate of vaccination, makes seed cell farthest be utilized; Secondly require there is higher kinetic rate, make seed cell have sufficient vigor; Finally to make attached cell high density and uniform distribution, to accelerate the speed of tissue regeneration and to improve the homogeneity of tissue. So it is required for optimizing cell vaccination ways and inoculation condition for the external tissue that successfully builds. Experiment is shown in cell and reaches 80% fusion and also can go down to posterity, at this moment transplant cells into and SIS cultivates, this is because Growth of Cells is often uneven when original cuiture, it is often that some regions have grown up to fine and close cellular layer, and other regions have not yet to see Growth of Cells, in this case, when whole bottle cell reaches and merges completely, fine and close cellular layer " aging ", functional status fails. Therefore, Secondary Culture is now namely carried out, it is possible to avoid local cells " aging ", activity to reduce, affect smooth muscle cell culture success ratio on SIS.
Smooth muscle cells inoculation density: due to the restriction of experiment condition, have employed traditional static vaccination ways and carries out cell inoculation. When inoculating due to static state, cell suspension is statically placed in material surface, part cell enters material internal along with liquid to material internal diffusion, but this passive introversive movement is limited, most cells is still deposited in material surface under gravity and causes that material surface has substantial amounts of cell adhesion, and private side is less. Experimentation finds, several milliliters are only had owing to inoculating suspension volume when static state is inoculated, obtain higher inoculum density, only it is improved the density of suspension, and high cell suspension density to be easily caused cell agglomerating, cause pore plugging, affect cell and enter material internal, simultaneously because the cellular binding sites of material surface is limited, a lot of cells are only loosely rest on surface, when adding culture medium, this part cell very easily leaves material and enters culture medium, and therefore rate of vaccination reduces along with the increase of inoculum density. Taking into full account the reason of these two aspects, if inoculum density is too low, seldom then rate of vaccination is low for the cell of entrance material internal, if inoculum density is too high, rate of vaccination also can reduce, and therefore to find a best inoculum density, can be only achieved maximum rate of vaccination. Author, through experiment screening, have selected 5.0 × 106/cm2Inoculum density, by check pathological section, result is ideal.
SISInterpretation as vagina smooth muscle cell carrier
Acellular matrix obtained after physics and chemical method process needs to verify whether the residual of cell component have a lot of methods can reach this purpose. The hematoxylin-eosin histological stain of standard is the line method whether having residual cell material in tissues observed.Under light microscopic, by many queueing disciplines, the webbed collagen fiber of compact siro spinning technology form SIS host material, and hematoxylin-eosin staining takes on a red color, and inside does not find cell debris. Show that SIS antigenicity is weak, it is possible to apply to the skin grafing and mending of tissue. SIS maintains good mechanical strength simultaneously, is suitable as the timbering material that vagina tissue engineering builds. The In vitro culture of seed cell and amplification are another key technologies of organizational project. The seed cell that vagina defect repair is commonly used includes the autologous smooth muscle cell of vagina and vaginal epithelium. Vagina must possess normal contraction and diastolic function as genitals, and these all rely on normal vagina smooth muscle cell and complete, and therefore vagina smooth muscle cell injuring model and amplification are the important foundations of organizational project vaginal reconstruction. Organizational project organ reparation needs cultivation and the amplification of cell in vitro, and a large amount of great-hearted seed cells are to ensure that the primary condition that organ well regenerates. This research In vitro culture finds that vagina smooth muscle cell can quickly go down to posterity after original cuiture, is suitable as seed cell and breeds in a large number in vitro. Cultivating cell to observe under inverted microscope, cell is fusiformis or polygon, and during fusion, smooth muscle cell characteristic growth perfonnance " peak-to-valley " shape structure occurs in subregion cell, meets smooth muscle cell growth form. Simultaneously vagina smooth muscle inoculate on SIS after well-grown, observed by dynamic hematoxylin-eosin staining and show that vagina smooth muscle cell is bred on the material and invades to the middle part of material. SABC identifies that the vagina smooth muscle cell SmoothMuscle α-actin being seeded on support expresses the positive, in Cytoplasm, a-actin is the neccessary composition of composition smooth muscle cell skeleton, it is the main structural protein of smooth muscle cell, for identifying the specific expressing protein of smooth muscle cell. This experiment institute cultured cells adopts a-actin immunohistochemical staining positive. Demonstrate the smooth muscle cell table shape being seeded on SIS material and have no change, remain to give expression to the cytoskeletal protein representing normal smooth muscle cell contraction, and can stick on SIS, grow, expand. Show the compatibility between smooth muscle cell and SIS good. Cell free SIS is that vagina smooth muscle cell provides attachment point from the results of view, its pore structure allows and can meet cellular metabolism and the requirement of nutrient substance discrepancy, additionally SIS is possibly together with multiple somatomedin, therefore smooth muscle cell can stick on SIS, grows, breeds, and do not destroy form and the organizational structure of cell.
Experimental system provides natural biologic bracket material for vagina tissue engineering research
The present invention utilizes pig SIS as the carrier of vagina smooth muscle Growth of Cells, to observe the smooth muscle cell after In vitro culture on carrier, its intrinsic biological characteristics and function can be maintained, and can cell-SIS complex to the differentiation of predetermined organizational structure and differentiation. A-Actin is measured through hematoxylin-eosin staining and immunohistochemical staining, observing that the smooth muscle cell transplantation after In vitro culture is after SIS, under the supporting role of pig SIS, cell still has the expression of a-actin, and stratification can be broken up, maintain the construction features of smooth muscle cell. These phenomenons clearly illustrate that the surface activity of SIS is suitable for the sticking of cell, grow, and its pore structure allows the requirement that also can meet cellular metabolism and nutrient substance discrepancy.
Conclusion: the smooth muscle cell of In vitro culture is after SIS kind is planted, detect through morphology and SABC, smooth muscle cell can attach on SIS, breaks up and breed, show that the cell compatibility of SIS is good, do not affect the form of smooth muscle cell, cell growth and functional expression also have no obvious inhibiting effect and abundance, cheap. Therefore it is feasible that SIS can be used as vagina tissue engineering material, provide experimental model for vagina tissue engineering scaffold material simultaneously.
Conclusion: 1. external successfully turn out vagina smooth muscle cell, under inverted microscope, is shown in that the vagina smooth muscle cell of cultivation presents long shuttle shape, and cell is assembled and formed typical " peak and valley " sample configuration on culture dish. 2. Submucosa acellular matrix white in appearance, translucent, has certain toughness. Hematoxylin-eosin staining has no cell component to be existed. 3., after vagina smooth muscle cell-intestinal mucosa lower floor specimen section hematoxylin-eosin staining, under light microscopic, visible cell composition increases gradually, is grown to deep layer position by table is shallow. 4. after vagina smooth muscle cell-intestinal mucosa lower floor specimen section adopts anti-rabbit Smooth muscle α-actin monoclonal antibody immunity histochemical staining, the positive cell of all visible anti-rabbit α-Actin. Result preliminary proof intestinal mucosa underlying substrate can as a kind of smooth muscle cell carrier.
The above is only the preferred embodiment of the present invention; it is noted that, for those skilled in the art; under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (6)
1. the method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix, comprises the following steps:
1)Prepared by SIS:
Physical method processes: small intestinal takes from weight > the family pig of 200kg; By small intestinal freezed storage in transportation, in 4h, complete cleaning process; Placenta percreta and the muscle layer of small intestinal is removed, therebetween persistently with 40 DEG C of warm water washings with the handle of a knife being wrapped with gauze;
Chemical method processes: cuts the small intestinal processed through physical method along the longitudinal axis, is cut into every section of 15cm length, prepares by Abraham method; Each step of chemical method processing procedure all at room temperature carries out, and the ratio that material amasss with solution is held at 1:100;
2)The separation and Culture of vagina smooth muscle cell: draw materials, separate: choose 2.0 ~ 2.5kg doe, after ketamine 25mg/kg intramuscular injection anesthesia, fixing limbs, fully expose cloacal aperture; Hypogastric region and external genital iodophor disinfection, vagina rinses with aseptic metronidazole liquid; Above pubic symphysis, do about 5cm otch along hunter's line, cut skin, rectus abdominis m. and peritoneum successively, mention vagina, it is seen that the vagina tissue being connected with double uterus, after completely cutting vagina tissue, sewing-up cut, successively closes abdomen; Repeatedly rinse, with physiological saline solution, dual anti-liquid, the vagina tissue taken off, specimen is put in the dual anti-liquid of the PBS+ being made ready beforehand for afterwards, be sent to laboratory; Vagina tissue block is soaked in 5min in 0.05% hibitane by superclean bench, again by the moistening piece of tissue of DMEM, carefully separating removal mucosal epithelium layer and serous coat connective tissue layer with blade and ophthalmic operating set, then tiling to prune by the smooth muscle tissue of separator well forms 1mm3Piece of tissue, and make piece of tissue neat in edge, smooth;
3)Original cuiture: piece of tissue+enzyme digestion carries out original cuiture;The piece of tissue pruned is digested 0.5 ~ 1.0h with 0.1% collagenase IV in 37 DEG C of incubators, digestion is stopped when becoming hair to piece of tissue edge, then the piece of tissue digested is inoculated on culture dish, interval is about 5mm, quiescent culture 3.0 ~ 4.0h in 37 DEG C of constant incubators, after tissue block adherent is firm, add containing the DMEM culture fluid that volume fraction is 10% hyclone again, 37 DEG C of constant temperature quiescent culture 3.0 ~ 4.0d, change liquid after cell is swum out of, and change weekly liquid twice or thrice to maintain Growth of Cells;
4)Vagina smooth muscle cell is planted on SIS:Go down to posterity when Growth of Cells reaches 80% fusion, discard old culture medium, mixture slaking liquid is added to culture bottle, hatch 3-7min for 37 DEG C, observe that intercellular substance increases, cell is rounded, add the hyclone that volume fraction is 0.8% when being partially suspended in Digestive system in culture dish and terminate digestion, by centrifugal for cell suspension 5min, centrifugal collecting cell, culture medium settling flux, with 5.0 × 106/cm2Be inoculated on SIS substrate inner face, 37 DEG C, volume fraction be 5%CO2Quiescent culture in incubator; Culture medium is still containing the DMEM culture fluid that volume fraction is 10% hyclone, changes culture medium once every 2d.
2. the method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix according to claim 1, it is characterised in that: chemical method processes and specifically refers to: (1) is at the middle immersion under 37 DEG C of conditions containing 0.5mmol/L ethylenediaminetetraacetic acid (EDTA) and 0.4% tryptic solution, stirring 5.0 ~ 6.0h; (2) continue, with 0.2% glutaraldehyde, tissue is carried out crosslinking to protect; Rinse well with deionized water, be placed in the solution containing 40 μm of ol/LDNA enzymes and 1mmol/L sodium chloride soak, stirring pig small intestine 6 ~ 8h; (3) with soaking in sodium chloride (1mmol/L) phosphate buffer (PBS) after deionized water rinsing, stirring 16h; (4) with soaking 2h after deionized water rinsing in PBS solution (pH value is 7.0 ~ 7.4); (5) again with deionized water rinsing substrate 2h; (6) sterilization: SIS is soaked in 20% alcoholic solution containing 0.1% peracetic acid 8h, with containing 0.05% Hydrazoic acid,sodium salt PBS solution cleaning 2h, can short-term preservation in 4 DEG C of PBS solution.
3. the method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix according to claim 2, it is characterised in that: chemical method processing procedure, except sterilization, carries out under all immersions in magnetic stirring apparatus, stirring in all of processing procedure.
4. the method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix according to claim 1, it is characterised in that: described dual anti-liquid is the mixed solution of penicillin and streptomycin.
5. the method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix according to claim 1, it is characterised in that: described mixture slaking liquid is 0.25% pancreatin: 0.02%EDTA=1:1.
6. the method preparing smooth muscle cell carrier based on small intestinal submucosa acellular matrix according to claim 1, it is characterised in that: described centrifugal rotational speed is 1000r/min, centrifugal radius 13cm.
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