CN106267346B - Method that is a kind of while handling a variety of biological tissues - Google Patents
Method that is a kind of while handling a variety of biological tissues Download PDFInfo
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- CN106267346B CN106267346B CN201610651580.XA CN201610651580A CN106267346B CN 106267346 B CN106267346 B CN 106267346B CN 201610651580 A CN201610651580 A CN 201610651580A CN 106267346 B CN106267346 B CN 106267346B
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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
- 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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- 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
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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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
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Abstract
The invention discloses a kind of methods for handling a variety of biological tissues simultaneously, comprising steps of pretreatment, degreasing, de- cell, crosslinking, sterilizing.The method that a variety of biological tissues are handled while proposed by the invention can handle a variety of biological tissues simultaneously, and batch processing biological tissue amount is big, and degree for the treatment of is uniform, simple process, is easy to industrialization, realize efficiently production.The method that a variety of biological tissues are handled while proposed by the invention avoids the native three dimensional collagen scaffold fibre structure that can retain a variety of biological tissue cell epimatrixs using strong acid, highly basic and protease completely, removes the cell component for causing immunogenicity in tissue.The biological tissue prepared using this method is had good biocompatibility and mechanical performance, and compares natural biological tissue, is had compared with highly resistance degradation capability, be can be used for all kinds of soft tissue healings.
Description
Technical field
The present invention relates to organizational engineering medical biomaterial technical fields, handle a variety of lifes simultaneously more particularly to a kind of
The method of object tissue.
Background technique
As social senilization's phenomenon is increasingly severe, cancer incidence is consequently increased.After all kinds of solid tumor excisions, often
The defect or weakness for often resulting in tumor tissues surrounding soft tissue cause chest wall soft tissue weak after mammary gland carcinectomy, then row cream
When room Reconstruction, often easily cause the complication such as prostheses migration, protrusion.
In view of the above-mentioned problems, after carrying out tumor resection using allogeneic acellular dermal matrix and xenogenesis acellular matrix
Soft tissue repair effect is preferable, and domestic and international clinical application has more than two decades history.Such acellular matrix product can also be used for
The enhancing or reparation of all kinds of soft tissue weakness caused by other reasons, such as all kinds of hernia reparations, tendon reinforcement, rotator cuff reparation, intestines
Stomach previous anastomotic reinforcing etc..
There is the more patented technology report for preparing acellular matrix at present, but since method is all made of strong acid, highly basic, albumen
Enzyme or surfactant etc. can not handle simultaneously a variety of different groups to the biggish reagent of disorganization with batch same process parameter
Structure, different-thickness, heterogeneity and the biological tissue in source are knitted, otherwise various biological tissues not can be implemented simultaneously preferable de-
Cell effect or preferable degradation property.Such as the pig dermis thicker, institutional framework is finer and close and relatively thin, institutional framework are loosely
Trees-Osima jacoti, Osima excavata carry out while handling, tend to realize the former completely remove cell and guarantee tissue be not damaged,
When the preferable technological parameter of degradation property is used for the latter, the latter can also completely remove cell, but disorganization is then larger, degradability
It can not can guarantee；And it can be realized the latter and completely remove cell and guarantee that tissue is not damaged, the preferable technique of degradation property is joined
When number is for the former, the former cannot achieve cell and completely removes, and not be able to satisfy Product Safety related request.In addition to this, mesh
Preceding patented technology report also has following defects:
(1) using enzyme and TritonX-100 as de- cell reagent, reagent remains more difficult cleaning, destroys to collagenous fibres
It is larger；
(2) it is sterilized using benzalkonium bromide solution, NaCl or EDTA hypertonic solution and detergent Tween, SDS or Triton are de-
Cell, finally using ethylene oxide sterilizing after phosphate buffer washing, the thimerosal that this method uses is more toxic, use
The de- more difficult cleaning of cell reagent destroys collagenous fibres also larger；
(3) using also use in technique Peracetic acid strong acid, detergent, pancreatin etc. to the biggish reagent of material damage,
Material integrity is destroyed.
(4) it uses in technique and uses the chemical cross-linking agent being more toxic, such as glutaraldehyde, epoxides, residual crosslinker
Biocompatibility is easily reduced, the material of partial cross-linked dose of processing, which implants, easily causes calcification.
(5) single batch is only capable of processing single biological tissue, and efficiency is lower, and industrial device utilization rate is low.
To solve the above-mentioned problems, the present invention needs to develop method that is a kind of while handling a variety of biological tissues.
Summary of the invention
The object of the present invention is to provide a kind of methods for handling a variety of biological tissues simultaneously.
The first aspect of the present invention provides method that is a kind of while handling a variety of biological tissues, comprising the following steps:
(a) it pre-processes: removing unwanted tissues after biological tissue is cleaned, and carry out disinfection, obtain pretreated target group
It knits；
(b) degreasing: the pretreated destination organization is impregnated using NaTDC or isopropanol, obtains the target of degreasing
Tissue；
(c) it takes off cell: being soaked by the destination organization that high-pressure physics method handles degreasing with smudge cells, then by DNA enzymatic
Bubble and α-Gal enzyme solutions impregnate all cell fragments of removal and antigen obtains cell free destination organization；
(d) it is crosslinked: the cell free destination organization being crosslinked using Physical cross linking methods or Chemical Crosslinking Methods.
(e) it sterilizes: being sterilized using irradiation or ethylene oxide to the destination organization being crosslinked through step (d).
In another preferred example, the biological tissue refers to animal sources or allohisto compatibility, including chitterlings, pigskin, ox
Two or more any biological group such as skin, bovine pericardium, Pigs Hearts packet, alloskin, cor bovinum valve, pig heart valve
It knits.
In another preferred example, the unwanted tissues are fat.
In another preferred example, in step a), the cleaning is to be cleaned using purified water.
In another preferred example, in step a), the disinfection is to be sterilized under 75% ethyl alcohol or 2% hydrogen peroxide at room temperature
0.5-2h。
In another preferred example, described to impregnate for the pretreated destination organization is placed in 1-3% deoxidation in step b)
12-48h is impregnated at room temperature in sodium taurocholate or isopropanol.
In another preferred example, it in step b), is rinsed 3-8 times with purified water again after immersion, removes NaTDC or different
Propyl alcohol.
In another preferred example, the high-pressure physics method refers to that the destination organization by the degreasing is placed in extra-high tension unit
In, 1-60min is handled under the conditions of 1-10MPa, 4-40 DEG C.
In another preferred example, the DNA enzymatic, which is impregnated, impregnates 4- for the DNA enzymatic solution oscillation at room temperature using 1-50U/mL
24h。
In another preferred example, the α-Gal enzyme solutions are impregnated to be shaken at room temperature using the α-Gal enzyme solutions of 1-50U/L
It swings and impregnates 4-24h.
In another preferred example, the frequency of oscillation that the oscillation is impregnated is 80-150rpm.
In another preferred example, the oscillation is cleaned 5-10 times after impregnating with purified water.
In another preferred example, the Physical cross linking methods refer to that the cell free destination organization, which is carried out freezing, to be done
It is dry, make its moisture content lower than 10%, then be placed in a vacuum drying oven, under the conditions of 80-130 DEG C, vacuum heat treatment 24-120h,
Then packaging send sterilizing.
In another preferred example, the Chemical Crosslinking Methods are that the cell free destination organization is soaked in 0.01-
In the EDC solution of 0.5mol/L, it is crosslinked 12-48h at room temperature, then cleaned 3-8 times with purified water, finally packaging send sterilizing.
In another preferred example, the irradiation bomb used that irradiates is gamma ray or electron beam, sterilizing dose 15-
30kGy。
In another preferred example, use the parameter of ethylene oxide sterilizing for the 4-20h that at 35-55 DEG C, sterilizes, ethylene oxide is dense
Spend 500-900mg/L.
The method that a variety of biological tissues are handled while proposed by the invention can handle a variety of biological tissues simultaneously, at batch
Manage biological tissue's amount greatly, degree for the treatment of is uniform, simple process, is easy to industrialization, realizes efficiently production.
The method that a variety of biological tissues are handled while proposed by the invention is avoided completely using strong acid, highly basic and albumen
Enzyme can retain the native three dimensional collagen scaffold fibre structure of a variety of biological tissue cell epimatrixs, remove and cause to be immunized in tissue
The cell component of originality.The biological tissue prepared using this method is had good biocompatibility and mechanical performance, and compared
Natural biological tissue has compared with highly resistance degradation capability, can be used for all kinds of soft tissue healings.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 is that the HE of a variety of biological tissues with batch processed dyes picture, and wherein A is acellular allodermis matrix, and B is
De- cell trees-Osima jacoti, Osima excavata matrix, C are de- cell bovine pericardium.
Fig. 2 is the acellular allodermis matrix with batch processed, takes off cell trees-Osima jacoti, Osima excavata matrix and de- cell ox
Pericardium before heat treatment after degradation property compare picture.
Fig. 3 is the acellular allodermis matrix, de- cell bovine pericardium, de- cell trees-Osima jacoti, Osima excavata with batch processed
Matrix and similar product degradation property compare picture.
Fig. 4 is the de- cell Pigs Hearts packet with batch processed, takes off the cell of cell ox dermal matrix, de- cell pig heart valve
Toxicity detection result picture.
Specific embodiment
The present inventor after extensive and in-depth study, develops side that is a kind of while handling a variety of biological tissues for the first time
Method.The method includes the steps: pretreatment, degreasing, physical method take off cell, crosslinking, irradiation sterilization.Proposed by the invention is same
When handle a variety of biological tissues method, a variety of biological tissues can be handled simultaneously, batch processing biological tissue amount is big, process journey
Uniform, simple process is spent, is easy to industrialization, realizes efficiently production, avoids industrial device from feeding intake less, relative energy consumption is big asks
Topic.
Processing method
The method that a variety of biological tissues are handled while of the invention, comprising steps of pretreatment, degreasing, physical method are de- thin
Born of the same parents, crosslinking, sterilizing.
Step 1: pretreatment
Different kind organism tissue is cleaned using purified water, removing unwanted tissues such as fat, and with 75% ethyl alcohol or 2% peroxide
Change hydrogen and sterilizes 0.5-2h at room temperature.Different kind organism tissue refers to animal sources or allohisto compatibility, including chitterlings, pigskin, ox-hide,
Two or more any biological tissue such as bovine pericardium, Pigs Hearts packet, alloskin, cor bovinum valve, pig heart valve.
For this step by cleaning, physics removes unwanted tissues, and carries out disinfection, and obtains pretreatment goal tissue so as to subsequent
Processing.
Step 2: degreasing
It will complete the pretreated various organization of step 1 and be placed in 1-3% NaTDC or isopropanol to impregnate at room temperature
12-48h is rinsed 3-8 times with purified water again after immersion, removes NaTDC or isopropanol.
This step is impregnated by NaTDC or isopropanol, obtains the destination organization of degreasing.
Due to this step degreasing technique to material structure and integrality without destruction, so for thickness difference, structure is different
Various Tissues handle simultaneously, select soaking time to be most difficult to the tissue of degreasing as standard.
Step 3: physical method takes off cell
Will complete step 2 degreasing various organization, be placed in extra-high tension unit, under the conditions of 1-10MPa, 4-40 DEG C at
1-60min is managed, then is vibrated at room temperature with 1-50U/mL DNA enzymatic solution and impregnates 4-24h, 1-50U/L α-Gal enzyme solutions are at room temperature
4-24h is impregnated in oscillation, and frequency of oscillation 80-150rpm is finally cleaned 5-10 times with purified water.
This step is handled by high-pressure physics method, it can be achieved that the various organization internal cells that thickness is different, structure is different
It is crushed, is not influenced by tissue-derived simultaneously；Again by DNA enzymatic and α-Gal enzyme solutions immersion can remove all cell fragments and
Antigen.Various tissue treatment degree are uniform controllable.The DNA enzymatic and α-Gal enzyme that this step uses are non-protein enzyme, to biological group
Structure and integrality are knitted without destruction.
Step 4: crosslinking
Physical cross linking methods can be used, be such as heat-treated: it is dry with batch freezing that the cell free various organization of step 3 will be completed
It is dry, make its moisture content lower than 10%, then be placed in a vacuum drying oven, under the conditions of 80-130 DEG C, vacuum heat treatment 24-120h,
Then packaging send sterilizing.
Chemical Crosslinking Methods can also be used, as EDC (carbodiimide) is handled: it is cell free all kinds of that step 3 will be completed
Tissue is soaked in the EDC solution of 0.01-0.5mol/L with batch, is crosslinked 12-48h at room temperature, then clean 3-8 with purified water
Secondary, finally packaging send sterilizing.
This step is handled by vacuum high-temperature physical method or using noresidue, the higher catalyst EDC of biocompatibility
Solution immersion treatment is not influenced, it can be achieved that the various tissues while crosslinking Treatment that thickness is different, structure is different by tissue-derived.
Various tissue treatment degree are uniform controllable.
Catalyst EDC has been used for the crosslinking Treatment of a variety of implanted medical device products such as collagem membrane, hyaluronic acid derivatives,
Amide reaction occurs for the carboxyl that can be catalyzed in tropocollagen molecule, does not change itself as catalyst, and be easy to be dissolved in
Water, residual can be completely removed by being cleaned with purified water.
Step 5: sterilizing
The various organization that final packaging is finished is sterilized using irradiation sterilization or ethylene oxide sterilizing mode；Irradiation
The sterilizing dose of sterilizing is 15-30kGy, and irradiation bomb is gamma ray or electron beam；Ethylene oxide sterilizing parameter is 35-55 DEG C
Under, sterilize 4-20h, ethylene oxide concentration 500-900mg/L.
This step is by irradiation or ethylene oxide sterilizing, it can be achieved that all various organizations reach germ-free condition, and can play
Inactivate the effect of each viroid.
The present invention is not that Alternative step simply sorts, but there is innovation to find out and be suitble to a variety of biological tissues while locating
The processing step and parameter of reason.And present invention preparation side uses the 1-10MPa super-pressure physical method processing of innovation, so that
The effect for going cell key process of Various Tissues is uniform.
Main advantages of the present invention include:
(1) method that a variety of biological tissues are handled while proposed by the invention is avoided completely using strong acid, highly basic and egg
White enzyme can retain the native three dimensional collagen scaffold fibre structure of a variety of biological tissue cell epimatrixs, remove and cause to exempt from tissue
The cell component of epidemic focus.The biological tissue prepared using this method has good biocompatibility and mechanical performance, and phase
Than natural biological tissue, has compared with highly resistance degradation capability, can be used for all kinds of soft tissue healings.
(2) method that a variety of biological tissues are handled while proposed by the invention, can with a variety of biological tissues of batch processed,
Compared to the method for a batch processed single biological tissue, it is greatly improved process efficiency, especially when certain biological tissue
In the case where negligible amounts, can mix Various Tissues processing, avoid industrial large-sized equipment inventory too low, cause energy waste and
The relatively high problem of equipment loss.
(3) present invention mainly takes off cell using physical method, it can be achieved that the same batch processed of a variety of biological tissues, compares chemistry
Reagent facture degree for the treatment of is more uniform controllable.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and
Number is weight percent and parts by weight.
It should be noted that in the claim and specification of this patent, such as first and second or the like relationship
Term is only used to distinguish one entity or operation from another entity or operation, without necessarily requiring or implying
There are any actual relationship or orders between these entities or operation.Moreover, the terms "include", "comprise" or its
Any other variant is intended to non-exclusive inclusion so that include the process, methods of a series of elements, article or
Equipment not only includes those elements, but also including other elements that are not explicitly listed, or further include for this process,
Method, article or the intrinsic element of equipment.In the absence of more restrictions, being wanted by what sentence " including one " limited
Element, it is not excluded that there is also other identical elements in the process, method, article or apparatus that includes the element.
Embodiment 1
Step 1: pretreatment
Chitterlings, bovine pericardium, pigskin are cleaned using purified water, remove the mesenterium and serous coat of unwanted tissues such as chitterlings
Layer, bovine pericardium appearance adhering fat particle, pigskin epidermis and subcutaneous layer of fat etc. obtain trees-Osima jacoti, Osima excavata, bovine pericardium, pig
Skin corium, and 0.5h is sterilized at room temperature with 75% ethyl alcohol.
Step 2: degreasing
It will complete the pretreated various organization of step 1 and be placed in isopropanol to impregnate 48h at room temperature, again with purifying after immersion
Water rinses 8 times, removes isopropanol.
Step 3: physical method takes off cell
The various organization of step 2 degreasing will be completed, is placed in extra-high tension unit, is handled under the conditions of 10MPa, 4 DEG C
60min, then 100rpm oscillation is impregnated for 24 hours at room temperature with 50U/mL DNA enzymatic solution, the α-Gal enzyme solutions of 50U/L are at room temperature
100rpm oscillation is impregnated for 24 hours, is finally cleaned 10 times with purified water.
Step 4: crosslinking
It will complete the cell free various organization of step 3 to be freeze-dried with batch, and make its moisture content lower than 10%, then be placed in
In vacuum oven, under the conditions of 130 DEG C, vacuum heat treatment 120h, then packaging send sterilizing.
Step 5: irradiation sterilization
The various organization that final packaging is finished, is sterilized using irradiation sterilization；The sterilizing dose of irradiation sterilization is
30kGy, irradiation bomb are gamma ray.
De- cell trees-Osima jacoti, Osima excavata matrix that the present embodiment is prepared, de- cell bovine pericardium, de- cell pig dermis
Matrix realizes good de- cell effect, and each institutional framework is without destruction.
Fig. 1 is using the HE coloration result of above-mentioned three kinds tissues, and wherein A is acellular allodermis matrix, and B is de- cell ox
Pericardium, C are de- cell trees-Osima jacoti, Osima excavata matrix, are as can be seen from the figure remained without any nucleus, and each tissue collagen
Fibre compact is without destruction.
Embodiment 2
Step 1: pretreatment
Chitterlings, bovine pericardium, pigskin are cleaned using purified water, remove the mesenterium and serous coat of unwanted tissues such as chitterlings
Layer, bovine pericardium appearance adhering fat particle, pigskin epidermis and subcutaneous layer of fat etc. obtain trees-Osima jacoti, Osima excavata, bovine pericardium, pig
Skin corium, and 1h is sterilized under 2% hydrogen peroxide at room temperature.
Step 2: degreasing
It will complete the pretreated various organization of step 1 and be placed in 1% NaTDC to impregnate 12h at room temperature, after immersion again
It is rinsed 3 times with purified water, removes NaTDC.
Step 3: physical method takes off cell
The various organization of step 2 degreasing will be completed, is placed in extra-high tension unit, is handled under the conditions of 5MPa, 40 DEG C
1min, then 18h, 30U/L α-Gal enzyme solutions 80rpm at room temperature are impregnated in 120rpm oscillation at room temperature with 30U/mL DNA enzymatic solution
20h is impregnated in oscillation, is finally cleaned 8 times with purified water.
Step 4: crosslinking
It will complete the cell free various organization of step 3 to be freeze-dried with batch, and make its moisture content lower than 10%, then be placed in
In vacuum oven, under the conditions of 120 DEG C, vacuum heat treatment 100h, then packaging send sterilizing.
Step 5: irradiation sterilization
The various organization that final packaging is finished is sterilized using irradiation sterilization mode；The sterilizing dose of irradiation sterilization
For 25kGy, irradiation bomb is electron beam.
De- cell trees-Osima jacoti, Osima excavata matrix that the present embodiment is prepared, de- cell bovine pericardium, de- cell pig dermis
Matrix realizes same batch physics thermal crosslinking treatment, and the external degradation rate respectively organized is considerably slower than not thermally treated control
Tissue.
Fig. 2 is that above-mentioned three kinds tissues manufactured in the present embodiment and the external degradation of not thermally treated control tissue compare knot
Fruit.Wherein A is acellular allodermis matrix manufactured in the present embodiment (TC ADM) and not thermally treated de- cell pig dermis base
The external degradation of matter (U ADM) as a result, B be de- cell trees-Osima jacoti, Osima excavata matrix (TC SIS) manufactured in the present embodiment with not
The external degradation of thermally treated de- cell trees-Osima jacoti, Osima excavata matrix (U SIS) is as a result, C is manufactured in the present embodiment de- thin
The external degradation of born of the same parents' bovine pericardium (TC ABP) and not thermally treated de- cell bovine pericardium (U ABP) is as a result, above-mentioned three comparisons
Figure abscissa is degradation time, and unit h, ordinate is liver mass surplus ratio, unit %.It can be seen from the figure that adopting
Good heat is reached on the basis of realizing that more complete structure retains with various tissues prepared by the method for the present embodiment
Cross-linking effect, degradation rate are much more slowly than not thermally treated each control tissue.
Embodiment 3
Step 1: pretreatment
Chitterlings, bovine pericardium, pigskin are cleaned using purified water, remove the mesenterium and serous coat of unwanted tissues such as chitterlings
Layer, bovine pericardium appearance adhering fat particle, pigskin epidermis and subcutaneous layer of fat etc. obtain trees-Osima jacoti, Osima excavata, bovine pericardium, pig
Skin corium, and with 75% ethanol disinfection 2h.
Step 2: degreasing
It will complete the pretreated various organization of step 1 and be placed in 3% NaTDC to impregnate at room temperature for 24 hours, after immersion again
It is rinsed 4 times with purified water, removes NaTDC.
Step 3: physical method takes off cell
The various organization of step 2 degreasing will be completed, is placed in extra-high tension unit, is handled under the conditions of 1MPa, 25 DEG C
30min, then 4h is impregnated in 150rpm oscillation at room temperature with 1U/mL DNA enzymatic solution, 150rpm's 1U/L α-Gal enzyme solutions shakes at room temperature
It swings and impregnates 4h, finally cleaned 5 times with purified water.
Step 4: crosslinking
It will complete the cell free various organization of step 3 to be soaked in the EDC solution of 0.01mol/L with batch, at room temperature
It is crosslinked 12h, then is cleaned 3 times with purified water, finally packaging send sterilizing.
Step 5: irradiation sterilization
The various organization that final packaging is finished is sterilized using ethylene oxide sterilizing mode, ethylene oxide sterilizing ginseng
Number is 55 DEG C, and sterilize 4h, ethylene oxide concentration 900mg/L.
All kinds of de- cell tissues that the present embodiment is prepared are being realized good de- cell effect simultaneously, can also maintained
More complete institutional framework and integrality carry out external degradation rate comparison with similar product the results show that prepared by the present embodiment
The degradation property of obtained all kinds of de- cell tissues is superior to or no worse than similar product.
Fig. 3 is the external degradation comparing result of above-mentioned three kinds tissues and similar product manufactured in the present embodiment.Wherein A is this
The acellular allodermis matrix (TC ADM) of embodiment preparation and the external drop of similar uncrosslinked acellular porcine dermal matrix commodity
Solution is as a result, B is crosslinked the external of de- cell bovine pericardium commodity with similar for de- cell bovine pericardium (TC ABP) manufactured in the present embodiment
Degradation results, C are that de- cell trees-Osima jacoti, Osima excavata matrix (TC SIS) manufactured in the present embodiment and similar be crosslinked take off cell pig
As a result, above-mentioned three comparison diagram abscissas are degradation time, unit h is indulged the external degradation of submucous layer of small intestine matrix commodity
Coordinate is liver mass surplus ratio, unit %.It can be seen from the figure that various groups prepared using the method for the present embodiment
It knits, while realizing preferably de- cell effect, has reached good heat cross-linking effect, degradation rate is better than similar uncrosslinked
Product or with it is similar crosslinked close.
Embodiment 4
Step 1: pretreatment
Pigs Hearts packet, ox-hide, pig heart valve are cleaned using purified water, removing unwanted tissues such as Pigs Hearts packet appearance adheres to rouge
Fat particle, ox-hide epidermis and subcutaneous layer of fat etc. obtain Pigs Hearts packet, ox skin corium, pig heart valve, and with 75% ethyl alcohol room temperature
Lower disinfection 1h.
Step 2: degreasing
It will complete the pretreated various organization of step 1 and be placed in isopropanol to impregnate 36h at room temperature, again with purifying after immersion
Water rinses 5 times, removes isopropanol.
Step 3: physical method takes off cell
The various organization of step 2 degreasing will be completed, is placed in extra-high tension unit, is handled under the conditions of 8MPa, 30 DEG C
15min, then 10h is impregnated in 110rpm oscillation at room temperature with 20U/mL DNA enzymatic solution, 40U/L α-Gal enzyme solutions are at room temperature
20h is impregnated in 140rpm oscillation, is finally cleaned 7 times with purified water.
Step 4: crosslinking
It will complete the cell free various organization of step 3 to be soaked in the EDC solution of 0.5mol/L with batch, at room temperature
It is crosslinked 48h, then is cleaned 8 times with purified water, finally packaging send sterilizing.
Step 5: irradiation sterilization
The various organization that final packaging is finished is sterilized using ethylene oxide sterilizing mode, and sterilizing parameter is 35 DEG C
Sterilize 20h, ethylene oxide concentration 500mg/L.
All kinds of de- cell tissues that the present embodiment is prepared, while effectively removing cell, heat cross-linking, it may have good
Good biocompatibility and vitro cytotoxicity.
Fig. 4 is the cell Proliferation quantity figure that tissue vitro cytotoxicity testing result is prepared in above-mentioned three kinds of the present embodiment
Piece.Wherein A is blank control group, and B is negative control group, and C is positive controls, and D is de- cell Pigs Hearts packet, and E is de- cell ox
Dermal matrix, F are de- cell pig heart valve.It can be seen from the figure that blank control group A, negative control group B and this implementation
The cell Proliferation quantity for three kinds of de- cell tissues that example is prepared is close, and cellular morphology is normal, illustrates prepared by the present embodiment
Three kinds of obtained de- cell tissues have good Cyto-compatibility in vitro.
Embodiment 5
The present embodiment is same as Example 1, only in step 1 " chitterlings, bovine pericardium, pigskin are cleaned using purified water,
Remove the mesenterium and placenta percreta, bovine pericardium appearance adhering fat particle, pigskin epidermis and subcutaneous rouge of unwanted tissues such as chitterlings
Fat layer etc. obtains trees-Osima jacoti, Osima excavata, bovine pericardium, pig dermis layer " it is changed to " by chitterlings, alloskin, pigskin, ox
Skin, cor bovinum valve are cleaned using purified water, remove the mesenterium of unwanted tissues such as chitterlings and the epidermis of placenta percreta, all kinds of skins
And subcutaneous layer of fat etc., obtain trees-Osima jacoti, Osima excavata, allogeneic dermis layer, pig dermis layer, ox skin corium, cor bovinum valve
Film ".
Embodiment 6
The present embodiment is same as Example 3, only Step 3: physical method, which takes off in cell, " will complete each of step 2 degreasing
Class loading is placed in extra-high tension unit, 1min is handled under the conditions of 1MPa, 25 DEG C, then at room temperature with 1U/mL DNA enzymatic solution
4h is impregnated in 150rpm oscillation, and 4h is impregnated in 150rpm oscillation to 1U/L α-Gal enzyme solutions at room temperature, is finally cleaned 5 times with purified water " more
Be changed to " various organization of step 2 degreasing will be completed, is placed in extra-high tension unit, handles 25min under the conditions of 6MPa, 10 DEG C,
With 15U/mL DNA enzymatic solution, 14h is impregnated in 125rpm oscillation at room temperature again, and 115rpm vibrates 25U/L α-Gal enzyme solutions at room temperature
10h is impregnated, is finally cleaned 4 times with purified water ".
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (13)
1. a kind of method for handling biological tissue simultaneously, which is characterized in that the described method comprises the following steps:
(a) it pre-processes: removing unwanted tissues after biological tissue is cleaned, and carry out disinfection, obtain pretreated destination organization；
(b) degreasing: the pretreated destination organization is impregnated using NaTDC or isopropanol, obtains the target group of degreasing
It knits；
(c) take off cell: by high-pressure physics method handle degreasing destination organization with smudge cells, then by DNA enzymatic immersion and
α-Gal enzyme solutions impregnate all cell fragments of removal and antigen obtains cell free destination organization；
(d) it is crosslinked: the cell free destination organization being crosslinked using Physical cross linking methods or Chemical Crosslinking Methods；
(e) it sterilizes: being sterilized using irradiation or ethylene oxide to the destination organization being crosslinked through step (d),
Wherein, it in the step b), is rinsed 3-8 times with purified water again after immersion, removes NaTDC or isopropanol；
In the step c), the high-pressure physics method refers to that the destination organization by the degreasing is placed in extra-high tension unit,
1-60min is handled under the conditions of 1-10MPa, 4-40 DEG C.
2. the method as described in claim 1, which is characterized in that the biological tissue refers to animal sources or allohisto compatibility, packet
Include chitterlings, pigskin, ox-hide, bovine pericardium, Pigs Hearts packet, alloskin, cor bovinum valve, any two kinds in pig heart valve
Or two or more biological tissues.
3. the method as described in claim 1, which is characterized in that in step a), the disinfection is with 75% ethyl alcohol or 2% peroxide
Change hydrogen and sterilizes 0.5-2h at room temperature.
4. the method as described in claim 1, which is characterized in that in step b), described impregnate is by the pretreated target
Tissue is placed in 1-3% NaTDC or isopropanol impregnates 12-48h at room temperature.
5. the method as described in claim 1, which is characterized in that the unwanted tissues are fat.
6. the method as described in claim 1, which is characterized in that the DNA enzymatic impregnates the DNA to use 1-50U/mL at room temperature
4-24h is impregnated in enzyme solutions oscillation.
7. the method as described in claim 1, which is characterized in that the α-Gal enzyme solutions impregnate to use 1-50U/L at room temperature
α-Gal enzyme solutions oscillation impregnate 4-24h.
8. the method as described in claim 1, which is characterized in that the Physical cross linking methods refer to the cell free target
Tissue is freeze-dried, and makes its moisture content lower than 10%, then be placed in a vacuum drying oven, under the conditions of 80-130 DEG C, vacuum
It is heat-treated 24-120h, then packaging send sterilizing.
9. the method as described in claim 1, which is characterized in that the Chemical Crosslinking Methods are by the cell free target group
It knits and is soaked in the EDC solution of 0.01-0.5mol/L, be crosslinked 12-48h at room temperature, then cleaned 3-8 times with purified water, finally
Packaging send sterilizing.
10. the method as described in claim 1, which is characterized in that the irradiation bomb used that irradiates is gamma ray or electronics
Beam, sterilizing dose 15-30kGy.
11. method according to claim 6 or 7, which is characterized in that the frequency of oscillation for vibrating immersion is 80-150rpm.
12. method according to claim 6 or 7, which is characterized in that the oscillation is cleaned 5-10 times after impregnating with purified water.
13. the method as described in claim 1, which is characterized in that the parameter for using ethylene oxide to sterilize is 35-55 DEG C
Under, sterilize 4-20h, ethylene oxide concentration 500-900mg/L.
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| CN114276974A (en) * | 2021-12-24 | 2022-04-05 | 上海理工大学 | Interstitial material for encapsulating cells and preparation method and application thereof |
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