CN107164298A - Supercritical fluid technique prepares the method that soft tissue removes cellular matrix - Google Patents
Supercritical fluid technique prepares the method that soft tissue removes cellular matrix Download PDFInfo
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- 238000012545 processing Methods 0.000 claims description 32
- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 17
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 14
- 230000001954 sterilising effect Effects 0.000 claims description 14
- 230000008569 process Effects 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
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- 150000001335 aliphatic alkanes Chemical class 0.000 claims 1
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- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
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- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
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- 238000001514 detection method Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0081—Purging biological preparations of unwanted cells
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- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
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Abstract
The present invention discloses the preparation method that a kind of soft tissue removes cellular matrix, it is related to clinical medicine, biomedical and regeneration medicine technology field, a kind of soft tissue that the present invention is provided goes the preparation method of cellular matrix, natural soft tissue, which is prepared, using supercritical fluid technique removes cellular matrix, wherein, natural soft tissue includes small intestine, vascular tissue, musculature, heart tissue, valvular tissue, lung tissue, spleen tissue, renal tissue, liver organization, gastric tissue, gallbladder tissue, adipose tissue, cartilaginous tissue, tracheal tissue, esophageal tissue, bladder body, urine output tubing, oviduct tissue or uterine tissue.The soft tissue that the present invention is provided goes the preparation method of cellular matrix, and the destruction to soft tissue matrix components is smaller, and the soft tissue of acquisition goes the immunogenicity of cellular matrix low, good biocompatibility.
Description
Technical field
The present invention relates to clinical medicine, biomedical and regeneration medicine technology field, more particularly, to a kind of shooting flow
Body technique prepares the method that soft tissue removes cellular matrix.
Background technology
It is to be carried out tissue/organ with appropriate method in Cell extraction, removing tissue to cause to remove cellular matrix
The cell component of rejection, keeps the three-dimensional structure of tissue/organ.Compared with the material of the non-animal such as metal, plastics,
Go the composition and structure of cellular matrix closer to tissue, biocompatibility is more excellent, and this makes organizational project reparation
In the material that is most widely used.The process for removing cell is exactly to be removed by methods such as physical method, chemical method and biology enzymes
Cell in tissue/organ, reduces the content of the immunogenic substances such as cell component therein as far as possible.Go the effective of cellular processes
Property depend on many factors, such as cell type, tissue density, thickness and lipid content.
Soft tissue is gone in cellular matrix, containing abundant collagen, aminoglycan etc., with good biocompatibility
And biomechanical property.A variety of soft tissues remove cellular matrix, such as corium go cellular matrix, small intestinal submucosa go cellular matrix,
Blood vessel removes cellular matrix etc., and the regeneration of the tissue defects such as skin, blood vessel, stomach wall is had been widely used in Tissue Engineering Study
Repair, and show good promotion organization power of regeneration.Therefore, by the cell component in soft tissue, make without thin
The soft tissue of born of the same parents removes cellular matrix, may be used as the biologic bracket material of suitable cell migration, growth and propagation.
Existing soft tissue goes to use the solution such as protease and lauryl sodium sulfate (SDS) cellular matrix preparation technology more
The cell in tissue is removed, but effect of these reagents to tissue is violent, destroys serious to matrix components, is easily caused tissue base
Matter is degraded.In addition, these reagents are difficult to remove, residual may cause cytotoxicity in the tissue, and biocompatibility is poor.
Traditional soft tissue goes cellular matrix preparation technology to be easily caused matrix components to be more easily damaged, and reagent is remained
Seriously, serious immunological rejection may be caused in implanting tissue.
Traditional soft tissue goes cellular matrix preparation technology to cause substrate degradation, mechanical properties decrease.
Traditional soft tissue goes cellular matrix preparation technology to need to take a substantial amount of time the residual cell removed in tissue
With the reagent of residual.
The content of the invention
It is an object of the invention to provide the preparation method that a kind of soft tissue removes cellular matrix, this method goes cell efficiency
Height, the destruction to the matrix components of soft tissue is smaller, while make its immunogenicity low, good biocompatibility.
In order to solve the above technical problems, the present invention is adopted the following technical scheme that：
A kind of soft tissue goes the preparation method of cellular matrix, and this method includes：Soft group is prepared using supercritical fluid technique
Cellular matrix is knitted, the soft tissue is natural soft tissue, and the natural soft tissue includes small intestine, vascular tissue, muscle
Tissue, heart tissue, valvular tissue, lung tissue, spleen tissue, renal tissue, liver organization, gastric tissue, gallbladder tissue, fat
Tissue, cartilaginous tissue, tracheal tissue, esophageal tissue, bladder body, urine output tubing, oviduct tissue or uterine tissue.
Preferably, this method specifically includes pretreatment, sterilization, goes cell, rinsing and sterilization steps.
Preferably, the pretreatment is under the conditions of 4-10 DEG C, to remove the impurity tissue in the soft tissue, cleans and treats
With.
Preferably, the sterilization be with a kind of vibration immersion in peracetic acid soln, ethanol solution and bromogeramine or
Vibration steeps for the soft tissue of pretreatment after several mixing, then is floated with physiological saline or phosphate buffer solution or deionized water
It is neutrality to be washed till pH.
Preferably, the concentration of the peracetic acid soln is 0.01-0.1wt%；The concentration of the ethanol solution is 1-
10%；The concentration of the bromogeramine is 0.05-1wt%；The soak time is 0.5-24h.Preferably, it is described go cell and
Rinsing uses supercritical CO2Fluid processing.
Preferably, the condition that the fluid for going cell step is handled is：The time of processing is 0.5-24h, the pressure of processing
It is 8-40MPa by force, the temperature of processing is 33-40 DEG C, and the pressure release speed after processing is 0.1-1MPa/min.
Preferably, methanol, absolute ethyl alcohol, trifluoroethanol and acetone are added in the fluid treatment process for going cell step
In one or more polar solvents as entrainer, the entrainer volume is 1-100 times of the soft tissue cumulative volume.
Preferably, the condition of the fluid processing of the rinse step is：The time of processing is 6-48h, and the pressure of processing is
8-40MPa, the temperature of processing is 33-40 DEG C, and the pressure release speed after processing is 0.1-1MPa/min；The fluid of the rinse step
Addition concentration is used as entrainer, entrainment for 0.5-10wt% tris solution or absolute ethyl alcohol in processing procedure
The volume of agent is 1-100 times of tissue volume.
Soft tissue prepared by the above method removes cellular matrix.
Beneficial effects of the present invention are as follows：
1. supercritical CO2Diffusion coefficient is big, and mass transfer velocity is fast, and soft tissue its content can be penetrated into a short time,
Several minutes or a few hours just can penetrate into soft tissue its content, and traditional chemical reagent usually requires 1 day even a couple of days
Soft tissue its content, the cell acted in tissue could be infiltrated into completely；
2.CO2With good Lipid dissolution performance, the lipid components in cell can be dissolved, the complete of cell membrane is destroyed
Property；
3. in pressure leak process, cell is because intracellular external differential is larger and drastically expands and ruptures when pressure declines, and release is thin
Intracellular is tolerant, is easy in subsequent treatment more thoroughly remove cellular content, the immunogenicity in reduction soft tissue matrix；
4. in pressure leak process, CO2Recovered by supercriticality to gaseous state, quickly escaped from soft tissue matrix, no
Easily remain in the tissue, it is to avoid chemical reagent is remained in soft tissue matrix, reduces the drift for removing chemical reagent in matrix
Wash the time, shorten the time cycle of Cell extraction；
5. supercritical CO2Soft tissue prepared by fluid technique removes cellular matrix, cell efficiency high is gone, to the matrix of soft tissue
The destruction of composition is smaller, and obtained soft tissue goes that cellular matrix immunogenicity is low, good biocompatibility.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The accompanying drawing used required in embodiment or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Figure 1A goes after cell to amplify 10 times of tissue staining figure for the intestinal mucosa lower floor prepared in embodiment 1；
Figure 1B goes after cell to amplify 20 times of tissue staining figure for the intestinal mucosa lower floor prepared in embodiment 1.
Embodiment
Technical scheme is clearly and completely described below in conjunction with accompanying drawing, it is clear that described implementation
Example is a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, ordinary skill
The every other embodiment that personnel are obtained under the premise of creative work is not made, belongs to the scope of protection of the invention.
Bio-derived material refers to a kind of biomaterial that natural biological tissue is formed after specially treated.In recent years make a return journey
Cell tissue matrix (Acellular Tissue Matrix) is more and more used for bio-derived material.
Traditional soft tissue goes cellular matrix preparation technology to be easily caused matrix components to be more easily damaged, and reagent is remained
Seriously, serious immunological rejection may be caused in implanting tissue.
Traditional soft tissue goes cellular matrix preparation technology to cause substrate degradation, mechanical properties decrease.
Traditional soft tissue goes cellular matrix preparation technology to need to take a substantial amount of time the residual cell removed in tissue
With the reagent of residual.
And supercritical CO2Diffusion coefficient is big, and mass transfer velocity is fast, and soft tissue its content can be penetrated into a short time,
Several minutes or a few hours just can penetrate into soft tissue its content, and traditional chemical reagent usually requires 1 day even a couple of days
Soft tissue its content, the cell acted in tissue could be infiltrated into completely；
CO2With good Lipid dissolution performance, the lipid components in cell can be dissolved, the complete of cell membrane is destroyed
Property；
In pressure leak process, cell discharges cell because intracellular external differential is larger and drastically expands and ruptures when pressure declines
Content, is easy in subsequent treatment more thoroughly remove cellular content, the immunogenicity in reduction soft tissue matrix；
In pressure leak process, CO2Recovered by supercriticality to gaseous state, quickly escaped from soft tissue matrix, will not
Residual is in the tissue, it is to avoid chemical reagent is remained in soft tissue matrix, reduces the rinsing for removing chemical reagent in matrix
Time, shorten the time cycle of Cell extraction.
So, supercritical fluid technique is removed cellular matrix by the present inventor for preparing soft tissue.
Soft tissue cells matrix is gone to comprise the following steps using supercritical fluid technique：
Cell, rinsing and sterilization steps are gone in pretreatment, sterilization.
In one preferred embodiment, pre-process under cryogenic, to remove other structural constituents in tissue,
Cleaned standby seam.
Another preferred embodiment in, sterilize for peracetic acid soln, ethanol solution or bromogeramine soak
A kind of vibration immersion or several mixing after vibration steep for the soft tissue of pretreatment, then it is neutral to be rinsed with water to pH；
The concentration of peracetic acid soln is preferably 0.01-0.1wt%；The concentration of ethanol solution is preferably 1-10%；It is new clean
The concentration preferably 0.05-1wt% that you go out；Soak time is preferably 0.5-24h.
Another preferred embodiment in, go cell to use supercritical CO2Fluid processing；
The condition for going the fluid of cell step to handle is：The time of processing is preferably 0.5-24h, and the pressure of processing is preferably
8-40MPa, the temperature of processing is preferably 33-40 DEG C, and the pressure release speed after processing is preferably 0.1-1MPa/min；
Go in the fluid treatment process of cell step to add one kind or many of methanol, absolute ethyl alcohol, trifluoroethanol and acetone
Plant as entrainer, the volume of the entrainer is 1-100 times of the soft tissue tissue volume.
Another preferred embodiment in, rinsing use supercritical CO2Fluid processing；
Rinse step fluid processing condition be：The time of processing is preferably 6-48h, and the pressure of processing is preferably 8-
40MPa, the temperature of processing is preferably 33-40 DEG C, and the pressure release speed after processing is preferably 0.1-1MPa/min；
In the fluid treatment process of rinse step add concentration be preferably 0.5-10wt% trishydroxymethylaminomethane or
Ethanol solution is as entrainer, and the volume of entrainer is 1-100 times of tissue volume.
Another preferred embodiment in, sterilized using gamma-ray irradiation.
Embodiment 1
Soft tissue is carried out using supercritical fluid technique to remove cell, by taking intestinal mucosa lower floor as an example, including following step
Suddenly：
Pretreatment：Chitterlings jejunal segment is taken, at low ambient temperatures, placenta percreta, muscle layer and mucous layer is struck off, floated with clear water
Clarification is washed till, intestinal mucosa lower floor is obtained；
Sterilization：The ethanol solution vibration immersion 2h that the peracetic acid soln and concentration for being 0.02wt% with concentration are 4%, is used
It is neutrality that clear water, which is rinsed to pH,；
Remove cell：Use supercritical CO2Fluid handles 2h, and pressure is 20MPa, and temperature is 37 DEG C, adds the work of absolute ethyl alcohol
For entrainer, the volume of absolute ethyl alcohol is 20 times of intestinal mucosa lower volume, and pressure release speed is 0.5MPa/min；
Rinsing：Use supercritical CO2Fluid handles 20h, and pressure is 20MPa, and temperature is 37 DEG C, and it is 2wt%'s to add concentration
Tris solution is as entrainer, and pressure release speed is 0.5MPa/min；
Sterilizing：Sterilized using gamma-ray irradiation, irradiation dose is 25kGy.
Embodiment 2
Soft tissue is carried out using supercritical fluid technique to remove cell, by taking intestinal mucosa lower floor as an example, including following step
Suddenly：
Pretreatment：Chitterlings jejunal segment is taken, at low ambient temperatures, placenta percreta, muscle layer and mucous layer is struck off, floated with clear water
Clarification is washed till, small intestinal submucosa is obtained；
Sterilization：The bromogeramine solution vibration that the peracetic acid soln or concentration for being 0.08wt% with concentration are 0.1wt%
0.5h is soaked, it is neutral to be rinsed with clear water to pH；
Remove cell：Use supercritical CO2Fluid handles 0.5h, and pressure is 30MPa, and temperature is 37 DEG C, add absolute ethyl alcohol and
The mixture of acetone is as entrainer, and the volume of the mixture of absolute ethyl alcohol and acetone is the 30 of intestinal mucosa lower volume
Times, pressure release speed is 0.2MPa/min；
Rinsing：Use supercritical CO2Fluid handles 15h, and pressure is 30MPa, and temperature is 37 DEG C, and addition concentration is 0.5wt%
Tris solution as entrainer, pressure release speed is 1MPa/min；
Sterilizing：Sterilized using x ray irradiation x, irradiation dose is 25kGy.
Embodiment 3
Intestinal mucosa lower floor is carried out using supercritical fluid technique to remove cell, comprised the following steps：
Pretreatment：Chitterlings jejunal segment is taken, at low ambient temperatures, placenta percreta, muscle layer and mucous layer is struck off, floated with clear water
Clarification is washed till, intestinal mucosa lower floor is obtained；
Sterilization：The ethanol solution vibration immersion 1h that the peracetic acid soln and concentration for being 0.04wt% with concentration are 5%, is used
It is neutrality that clear water, which is rinsed to pH,；
Remove cell：Use supercritical CO2Fluid handles 3.5h, and pressure is 10MPa, and temperature is 37 DEG C, add absolute ethyl alcohol and
The mixture of acetone is as entrainer, and the volume of the mixture of absolute ethyl alcohol and acetone is the 10 of intestinal mucosa lower volume
Times, pressure release speed is 1MPa/min；
Rinsing：Use supercritical CO2Fluid handles 12h, and pressure is 10MPa, and temperature is 37 DEG C, adds the second that concentration is 4%
Alcoholic solution is as entrainer, and pressure release speed is 0.2MPa/min；
Sterilizing：Sterilized using radiated by gamma-ray, irradiation dose is 25kGy.
Embodiment 4
HE dyeing is carried out to the intestinal mucosa matrix before and after Cell extraction in embodiment 1, as shown in Figure 1A and 1B, figure
1A is 10 times of figures of amplification, and Figure 1B is 20 times of figures of amplification, and coloration result shows, the intestinal mucosa lower floor after being handled in embodiment 1
In be substantially not visible cell, illustrate that the technique can effectively remove the cell in intestinal mucosa underlying substrate, reduce matrix
Immunogenicity.
Embodiment 5
With reference to national standard YY/T 0606.25-2014 to the chitterlings before and after embodiment 1 to the Cell extraction of embodiment 3
Mucous membrane matrix has carried out DNA residues detections, through it is above-mentioned go cell PROCESS FOR TREATMENT after, the DNA in intestinal mucosa underlying substrate
Residual quantity is 41.89ng/mg, goes DNA residuals in cell matrix materials to be less than 50ng/mg.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
The present invention is described in detail with reference to foregoing embodiments for pipe, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing embodiments can so be modified, or which part or all technical characteristic are entered
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme.
Claims (10)
1. a kind of soft tissue goes the preparation method of cellular matrix, it is characterised in that this method includes：Use supercritical fluid technique
Prepare soft tissue and remove cellular matrix, the soft tissue is natural soft tissue, and the natural soft tissue includes small intestine, blood vessel group
Knit, musculature, heart tissue, valvular tissue, lung tissue, spleen tissue, renal tissue, liver organization, gastric tissue, gall-bladder group
Knit, adipose tissue, cartilaginous tissue, tracheal tissue, esophageal tissue, bladder body, urine output tubing, oviduct tissue or uterus group
Knit.
2. preparation method according to claim 1, it is characterised in that this method specifically includes pretreatment, sterilization, go thin
Born of the same parents, rinsing and sterilization steps.
3. preparation method according to claim 2, it is characterised in that the pretreatment is under the conditions of 4-10 DEG C, to remove
Impurity tissue in the soft tissue, cleaned standby seam.
4. preparation method according to claim 2, it is characterised in that the sterilization is molten with peracetic acid soln, ethanol
Vibration steeps for the soft tissue of pretreatment after a kind of vibration immersion or several mixing in liquid and bromogeramine, then uses physiology salt
It is neutrality that water or phosphate buffer solution or deionized water, which are rinsed to pH,.
5. preparation method according to claim 4, it is characterised in that the concentration of the peracetic acid soln is 0.01-
0.1wt%；The concentration of the ethanol solution is 1-10%；The concentration of the bromogeramine is 0.05-1wt%；During the immersion
Between be 0.5-24h.
6. preparation method according to claim 2, it is characterised in that described to go cell and rinsing to use supercritical CO2Stream
Body processing.
7. preparation method according to claim 6, it is characterised in that the condition that the fluid for going cell step is handled
For：The time of processing is 0.5-24h, and the pressure of processing is 8-40MPa, and the temperature of processing is 33-40 DEG C, the pressure release speed after processing
Spend for 0.1-1MPa/min.
8. preparation method according to claim 6, it is characterised in that in the fluid treatment process for going cell step plus
Enter one or more polar solvents in methanol, absolute ethyl alcohol, trifluoroethanol and acetone as entrainer, the entrainer volume
For 1-100 times of the soft tissue cumulative volume.
9. preparation method according to claim 6, it is characterised in that the condition of the fluid processing of the rinse step is：
The time of processing is 6-48h, and the pressure of processing is 8-40MPa, and the temperature of processing is 33-40 DEG C, and the pressure release speed after processing is
0.1-1MPa/min；The trihydroxy methyl amino first that concentration is 0.5-10wt% is added in the fluid treatment process of the rinse step
Alkane solution or absolute ethyl alcohol are as entrainer, and the volume of entrainer is 1-100 times of tissue volume.
10. soft tissue prepared by the method as described in any one of claim 1 to 9 removes cellular matrix.
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| CN201710412419.1A CN107164298A (en) | 2017-06-02 | 2017-06-02 | Supercritical fluid technique prepares the method that soft tissue removes cellular matrix |
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| CN201710412419.1A CN107164298A (en) | 2017-06-02 | 2017-06-02 | Supercritical fluid technique prepares the method that soft tissue removes cellular matrix |
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| CN111068096A (en) * | 2019-12-24 | 2020-04-28 | 华侨大学 | A kind of preparation method of dECM-based wound repairing dressing |
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| CN113365672A (en) * | 2018-11-28 | 2021-09-07 | 株式会社多富 | Method for extracting extracellular matrix using supercritical fluid and extracellular matrix biomaterial for tissue regeneration produced thereby |
| CN113368313A (en) * | 2021-06-10 | 2021-09-10 | 吾奇生物医疗科技(江苏)有限公司 | Preparation method of biological membrane, product and application thereof |
| CN114286694A (en) * | 2019-08-27 | 2022-04-05 | 株式会社多富 | Animal adipose tissue-derived extracellular matrix and animal adipose tissue-derived extracellular matrix preservation solution |
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