CN109847096A - A kind of adipose tissue and preparation method thereof for tissue transplantation - Google Patents
A kind of adipose tissue and preparation method thereof for tissue transplantation Download PDFInfo
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Abstract
The adipose tissue and preparation method thereof that the present invention provides a kind of for tissue transplantation, is formed by the way that seed cell to be inoculated into biological support, space structure can be formed, prevent adipose tissue from implanting after liquefy；Cell growth helping matter is added simultaneously, the proliferation and update of adipose tissue implant inner posterior quadrant can be promoted, to accelerate tissue update and wound healing；Porcine HGF is added in secondary culture base, the division and update of mescenchymal stem cell can be effectively facilitated, improve the speed and quality of culture；To guarantee that induced medium and cell growth helping matter solution can be fully penetrated into space between cells, it is ultrasonically treated in advance, so that liquid is dispersed into droplet, improves its permeability.By above-mentioned means, the rate and the survival rate after transplanting that adipose tissue culture can be improved, to improve the success rate of adipose tissue transplantation.
Description
Technical field
The invention belongs to tissue engineering technique field, in particular to a kind of adipose tissue and its preparation for tissue transplantation
Method.
Background technique
Tissue transplantation is a certain position that autologous tissue or artificial material etc. are transplanted to body, to restore due to congenital
Or deformity or a kind of technology of tissue defect caused by day after tomorrow sexual factor.Tissue transplantation common at present includes dermatoplasty, rouge
Fat tissue transplantation, bone tissue transplanting etc., wherein adipose tissue transplantation also has in addition to therapeutic effect for patient with breast cancer
Important beauty function.Traditional solution is autograft, although satisfactory effect can be obtained, it is to sacrifice
Self health tissues are cost, will lead to many complication and additional injury while injuring original health tissues.Therefore mesh
The filling of preceding bioartificial materials becomes popular alternative.Bioartificial materials are by the way that histocyte to be inoculated into manually
The obtained culture of in vitro culture is carried out on material support, have physiological activity is good, acquisition histocyte quantity is few, specific aim and
The advantages that strong flexibility, and if cultivated using autologous patient histocyte, it can also be by the rejection after transplanting
It is preferably minimized, thus the burden for greatly improving the safety of transplanting, reducing patient.And improve the rate and shifting of adipose tissue culture
Survival rate after plant, and an important factor for raising adipose tissue transplantation success rate.
Summary of the invention
In order to solve the above-mentioned technical problems, the present invention provides a kind of for the adipose tissue of tissue transplantation and its preparation side
Method.
Specific technical solution of the present invention is as follows:
One aspect of the present invention provide a kind of adipose tissue for tissue transplantation, including biological support, fat cell with
And cell growth helping matter, the fat cell are inoculated into culture in the biological support by seed cell and obtain, described kind
Daughter cell is mescenchymal stem cell；
The biological support include Type I collagen, chondroitin sulfate, chitosan, aminoglucan, polylactic acid, polyorthoester,
At least one of poly-epsilon-caprolactone and poly lactide-glycolide acid；
Biological support should have good biological degradability and absorbability, good bio-compatibility, high porosity and suitable
In pore morphology, the characteristics such as matched structural strength, above-mentioned material are able to satisfy above-mentioned by force with implant site organization mechanics performance
Characteristic, and nonhazardous effect, safety are good, are suitable for being applied in biological support；
The cell growth helping matter includes Butyrospermum parkii fruit rouge, asiaticosid, hydrolysis of pea albumen, creatine, allantois
At least one of element and trehalose；
The appreciation rate that above-mentioned substance can promote cell metabolism, improve cell, to remarkably promote tissue growth and wound
Healing.
Further, the number that the seed cell is inoculated in every gram of biological support is 106~107It is a.
Further, the biological support includes the polyorthoester and polylactic acid-glycolic base that weight fraction ratio is 1:1~4
Acetate multipolymer.
Polyorthoester is that polynary ortho acid or polynary ortho esters and polyalcohols through being condensed in anhydrous conditions to form ortho esters
The product that key obtains, it is not soluble in water, it is not also swollen, can degrade from surface layer-by-layer, without being fragmented into aqueous solution
Fritter；Poly lactide-glycolide acid is polymerized at random by lactic acid and hydroxyacetic acid, have good biocompatibility and
Biological degradability, it is nontoxic, and the performance with good encystation and film forming；Biological support made of raw material using said ratio
It is functional, it can satisfy and use needs.
Further, the cell growth helping matter includes asiaticosid, the flesh that weight fraction ratio is 5:2~4:1~3
Acid and allantoin.
Asiaticosid can accelerate cell metabolism and division, promote tissue growth and wound healing；Creatine can be cell
Metabolism quickly provides energy, promotes cell growth and update；Allantoin can promote cell growth, cutin-softening albumen, thus
Accelerate tissue update and wound healing；Use the raw material of said ratio as cell growth helping matter, fatty group can be promoted
The update knitted, so that improving adipose tissue transplantation enters the growth rate after human body.
Another aspect of the present invention provides a kind of method for preparing above-mentioned adipose tissue, includes the following steps:
S1: extraction mescenchymal stem cell, originally culture 2~3 days, and obtained cell is carried out secondary culture 1~2 week；
S2: the cell that step S1 is obtained is mixed in proportion with biological support, and addition first passes through the induction of ultrasonic treatment in advance
Culture medium, gently oscillation mixes, and is placed in microgravity rotating generator and cultivates 1~2 week；
S3: to the obtained culture low-speed centrifugal of step S2 and discarding supernatant, and adds cell growth helping matter to get arriving
The adipose tissue.
Further, the step S1 includes the following steps:
S1.1: mescenchymal stem cell is extracted, is washed with 30~45min of collagenase digesting, low-speed centrifugal and with PBS buffer solution
It 2 times, by obtained cell inoculation into 10ml primary culture medium, is placed in 5%CO2, cultivated in 37 DEG C of incubator；
S1.2: when cell fusion degree reaches 80~90%, adherent cell collecting is thin by 1000~6000 after digestion
Born of the same parents/cm2 inoculum concentration is inoculated into 10ml secondary culture base, is placed in 5%CO2, is cultivated in 37 DEG C of incubator, every 3 days
It changes the liquid once, cultivates until when cell fusion degree reaches 80~90%, be centrifuged, discard culture solution, and washed with PBS buffer solution
2~3 times.
It will inoculation when often adulterating other a small amount of cells directly from the mescenchymal stem cell of tissue extraction, therefore cultivating
Amount reduces, and keeps the content of other cells of doping lower, so that the mesenchyma for inhibiting the proliferation of other cells, raising to obtain is dry thin
The purity of born of the same parents clone.
Further, the primary culture medium is that the DMEM-F12 of the fetal calf serum added with volume fraction 10~15% is trained
Nutrient solution；The secondary culture base is the DMEM-F12 training of the dense element of mandarin orange of growth factor and 5mg/L added with 50~100mg/L
Nutrient solution, the growth factor include the EGE and bFGF that weight fraction ratio is 1:1~1.5.
The dense element of mandarin orange is a kind of natural mixture extracted from orange peel, has the function of extremely strong antibacterial, can be inhibited thin
The growing multiplication of bacterium etc., and have no toxic side effect, the growth of cell will not be impacted, pathogenic microorganism will not be made to generate
Drug resistance.
EGE is also known as people oligopeptides -1, can promote cell proliferation differentiation, improve cell renewal rate；BFGF is important shape
State occurs and the inducible factor of differentiation, has and promotes angiogenic growth, promote wound healing and tissue repair, participate in nerve regneration etc.
Effect, and can play a role at low concentration (1mg/mL) in vitro；Using the growth factor of said ratio, Ke Yiyou
Effect promotes the division and update of mescenchymal stem cell, improves the speed and quality of culture.
Further, the step S2 includes the following steps:
S2.1: the cell and the biological support that step S1 is obtained are by 106~107A cell/g biological support ratio
Mixing；
S2.2: configuration induced medium, and 5~10min of sonic oscillation under the conditions of 25 DEG C, 40~100Hz, ultrasound terminate
Add in the mixed system that is obtained to step S2.1 of induced medium described in 5~10mL immediately afterwards, gently oscillation mix 1~
3min；The induced medium be Indomethacin added with 10~20mg/L, the vitamin C of 20~50mg/L and 10~
The DMEM-F12 culture solution of the sodium β-glycerophosphate of 20mg/L；
By ultrasonic treatment, make to be dispersed into droplet inside induced medium, to improve culture medium between cell
Permeability；The physical aspect that liquid is only temporarily changed due to being ultrasonically treated, should be immediately by induction after ultrasound
Culture medium is added in cell, to guarantee its permeability；
S2.3: the mixed system that step S2.2 is obtained is added in microgravity rotating generator, stands 30~45min, rotation
30~45min is stood again after turning 5~10min, repeats 3~4 times, rotating and culturing is started 1~2 week with the revolving speed of 15r/min, every 2
It empties the bubble generated during rotating and culturing, every 3~4 days one subcultures of replacement.
Further, in the step S3, the method for adding the cell growth helping matter is as follows:
The cell growth helping matter is added in plant source recombination human serum albumin in 2~8% ratio and is mixed
It closes uniformly, 5~10min of sonic oscillation under the conditions of 25 DEG C, 40~100Hz, adds 2~10ml immediately to step after ultrasound
In the culture that S2 is obtained, gently oscillation mixes 1~3min.
The physical aspect that liquid is only temporarily changed due to being ultrasonically treated, should be immediately by cell after ultrasound
The solution of growth helping matter is added in cell, to guarantee its permeability；And after the solution of cell growth helping matter is added
It also needs to be vibrated, to penetrate into solution further between cell.
Plant source recombination human serum albumin based on plant production manufacture, be capable of providing with the comparable nutritional ingredient of serum,
Fetal calf serum can be substituted, to reduce cost while providing sufficient nutrition for cell.
Beneficial effects of the present invention are as follows: the present invention provides a kind of for the adipose tissue of tissue transplantation and its preparation side
Method is formed by the way that seed cell to be inoculated into biological support, after can forming space structure, preventing adipose tissue from implanting
It liquefies；Cell growth helping matter is added simultaneously, the proliferation and update of adipose tissue implant inner posterior quadrant can be promoted,
To accelerate tissue update and wound healing；Porcine HGF is added in secondary culture base, it is dry that mesenchyma can be effectively facilitated
The division and update of cell, improve the speed and quality of culture；To guarantee induced medium and cell growth helping matter solution
Can be fully penetrated into space between cells, it is ultrasonically treated in advance, so that liquid is dispersed into droplet, improves its infiltration
Permeability.By above-mentioned means, the rate and the survival rate after transplanting that adipose tissue culture can be improved, to improve adipose tissue
The success rate of transplanting.
Specific embodiment
The preparation of main agents and culture medium
A.DMEM culture medium: specific ingredient is as follows:
| Serial number | Compound name | Content (mg/L) | Serial number | Compound name | Content (mg/L) |
| 1 | Anhydrous calcium chloride | 265.00 | 18 | Serine | 42.00 |
| 2 | Ferric nitrate | 0.10 | 19 | L-threonine | 95.00 |
| 3 | Potassium chloride | 400.00 | 20 | L-Trp | 16.00 |
| 4 | Anhydrous magnesium sulfate | 97.67 | 21 | L-tyrosine | 72.00 |
| 5 | Sodium chloride | 6400.00 | 22 | Valine | 94.00 |
| 6 | Anhydrous sodium dihydrogen phosphate | 109.00 | 23 | D-VB5 calcium | 4.00 |
| 7 | Succinic acid | 75.00 | 24 | Choline tartrate | 7.20 |
| 8 | Sodium succinate | 100.00 | 25 | Folic acid | 4.00 |
| 9 | L- R-gene | 84.00 | 26 | Inositol | 7.20 |
| 10 | L- hydrochloric acid cystine | 63.00 | 27 | Niacinamide | 4.00 |
| 11 | Glycine | 30.00 | 28 | Riboflavin | 0.40 |
| 12 | L- histidine monohydrochloride | 42.00 | 29 | Thiamine hydrochloride | 4.00 |
| 13 | L-Isoleucine | 105.00 | 30 | Pyridoxine hydrochloride | 4.00 |
| 14 | L-Leu | 105.00 | 31 | Glucose | 1000.00 |
| 15 | L-Lysine hydrochloride | 146.00 | 32 | Sodium Pyruvate | 110.00 |
| 16 | L-methionine | 30.00 | 33 | It is phenol red | 9.30.00 |
| 17 | L-phenylalanine | 66.00 |
B.PBS buffer:
Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4With 0.24g KH2PO4, it is dissolved in 800mL distilled water, uses
HCl adjusts the pH value of solution to 7.4, and finally plus distilled water is settled to 1L.In 15lbf/in2 (1034 × 105Pa) high pressure
Lower steam sterilization (at least 20min) is stored in room temperature or 4 DEG C of refrigerators.
Below with reference to following embodiment, invention is further described in detail.
Embodiment 1
A kind of adipose tissue for tissue transplantation, including biological support, mescenchymal stem cell and cell growth promote
Substance, the number that the mescenchymal stem cell is inoculated in every gram of biological support is 106~107It is a；The biological support includes
Weight fraction ratio is the polyorthoester and poly lactide-glycolide acid of 1:1；The cell growth helping matter includes weight
Measure asiaticosid, creatine and the allantoin that portion rate is 5:4:1.
Embodiment 2
A kind of adipose tissue for tissue transplantation, including biological support, mescenchymal stem cell and cell growth promote
Substance, the number that the mescenchymal stem cell is inoculated in every gram of biological support is 106~107It is a；The biological support includes
Weight fraction ratio is the polyorthoester and poly lactide-glycolide acid of 1:5；The cell growth helping matter includes weight
Measure asiaticosid, creatine and the allantoin that portion rate is 5:2:1.
Embodiment 3
A method of the adipose tissue for tissue transplantation is prepared, is included the following steps:
S1: extraction mescenchymal stem cell, originally culture 2~3 days, and obtained cell is carried out secondary culture 1~2 week；
S2: the cell that step S1 is obtained is mixed in proportion with biological support, and addition first passes through the induction of ultrasonic treatment in advance
Culture medium, gently oscillation mixes, and is placed in microgravity rotating generator and cultivates 1~2 week；
S3: to the obtained culture low-speed centrifugal of step S2 and discarding supernatant, and adds cell growth helping matter to get arriving
The adipose tissue.
Embodiment 4
A method of the adipose tissue for tissue transplantation, including each step described in embodiment 3 being prepared, wherein step S1
The specific method is as follows:
S1.1: extracting mescenchymal stem cell, wash 2 times with collagenase digesting 30min, low-speed centrifugal and with PBS buffer solution,
By obtained cell inoculation into 10ml primary culture medium, it is placed in 5%CO2, cultivated in 37 DEG C of incubator；It is described primary
Culture medium is the DMEM-F12 culture solution of the fetal calf serum added with volume fraction 10%
S1.2: when cell fusion degree reaches 80~90%, adherent cell collecting is thin by 1000~6000 after digestion
Born of the same parents/cm2Inoculum concentration be inoculated into 10ml secondary culture base, be placed in 5%CO2, cultivated in 37 DEG C of incubator, change within every 3 days
Liquid is primary, cultivates until when cell fusion degree reaches 80~90%, is centrifuged, discards culture solution, and wash 2 with PBS buffer solution
~3 times；The secondary culture base is the DMEM-F12 culture of the dense element of mandarin orange of growth factor and 5mg/L added with 50mg/L
Liquid, the growth factor include the EGE and bFGF that weight fraction ratio is 1:1.
Embodiment 5
A method of the adipose tissue for tissue transplantation, including each step described in embodiment 3 being prepared, wherein step S1
The specific method is as follows:
S1.1: extracting mescenchymal stem cell, wash 2 times with collagenase digesting 45min, low-speed centrifugal and with PBS buffer solution,
By obtained cell inoculation into 10ml primary culture medium, it is placed in 5%CO2, cultivated in 37 DEG C of incubator；It is described primary
Culture medium is the DMEM-F12 culture solution of the fetal calf serum added with volume fraction 15%
S1.2: when cell fusion degree reaches 80~90%, adherent cell collecting is thin by 1000~6000 after digestion
Born of the same parents/cm2Inoculum concentration be inoculated into 10ml secondary culture base, be placed in 5%CO2, cultivated in 37 DEG C of incubator, every 3 days
It changes the liquid once, cultivates until when cell fusion degree reaches 80~90%, be centrifuged, discard culture solution, and washed with PBS buffer solution
2~3 times；The secondary culture base is the DMEM-F12 culture of the dense element of mandarin orange of growth factor and 5mg/L added with 100mg/L
Liquid, the growth factor include the EGE and bFGF that weight fraction ratio is 1:1.5.
Embodiment 6
A method of the adipose tissue for tissue transplantation, including each step described in embodiment 4 being prepared, wherein step S2
The specific method is as follows:
S2.1: the cell and the biological support that step S1 is obtained are by 106~107A cell/g biological support ratio
Mixing；
S2.2: configuration induced medium, and sonic oscillation 10min under the conditions of 25 DEG C, 40Hz add immediately after ultrasonic
In the mixed system for adding induced medium described in 10ml to obtain to step S2.1, gently oscillation mixes 1min；The Fiber differentiation
Base is the vitamin C of Indomethacin, 20mg/L added with 20mg/L and the DMEM-F12 of the sodium β-glycerophosphate of 20mg/L
Culture solution；
S2.3: the mixed system that step S2.2 is obtained is added in microgravity rotating generator, stands 30min, rotation
30min is stood after 10min again, is repeated 3 times, rotating and culturing is started 1~2 week with the revolving speed of 15r/min, emptying in every 2 days is rotating
The bubble generated in incubation, every 3~4 days one subcultures of replacement.
Embodiment 7
A method of the adipose tissue for tissue transplantation, including each step described in embodiment 5 being prepared, wherein step S2
The specific method is as follows:
S2.1: the cell and the biological support that step S1 is obtained are by 106~107A cell/g biological support ratio
Mixing；
S2.2: configuration induced medium, and sonic oscillation 5min under the conditions of 25 DEG C, 100Hz add immediately after ultrasonic
In the mixed system for adding induced medium described in 10mL to obtain to step S2.1, gently oscillation mixes 3min；The Fiber differentiation
Base is the vitamin C of Indomethacin, 50mg/L added with 10mg/L and the DMEM-F12 of the sodium β-glycerophosphate of 10mg/L
Culture solution；
S2.3: the mixed system that step S2.2 is obtained is added in microgravity rotating generator, stands 45min, rotation
45min is stood after 5min again, is repeated 4 times, rotating and culturing is started 1~2 week with the revolving speed of 15r/min, emptying in every 2 days is trained in rotation
The bubble generated during supporting, every 3~4 days one subcultures of replacement.
Embodiment 8
A method of the adipose tissue for tissue transplantation, including each step described in embodiment 6 being prepared, wherein step S3
The method of middle addition cell growth helping matter is as follows:
Cell growth helping matter is added in plant source recombination human serum albumin in 2% ratio and is uniformly mixed,
Sonic oscillation 10min under the conditions of 25 DEG C, 40Hz is added in the culture that 10ml is obtained to step S2 immediately after ultrasonic,
Gently oscillation mixes 1min.
Embodiment 9
A method of the adipose tissue for tissue transplantation, including each step described in embodiment 7 being prepared, wherein step S3
The method of middle addition cell growth helping matter is as follows:
Cell growth helping matter is added in plant source recombination human serum albumin in 8% ratio and is uniformly mixed,
Sonic oscillation 5min under the conditions of 25 DEG C, 100Hz is added in the culture that 2mL is obtained to step S2, gently immediately after ultrasonic
Light oscillation mixes 3min.
Reference examples 1
A method of the adipose tissue for tissue transplantation, including each step described in embodiment 4 being prepared, wherein step S2
The specific method is as follows:
S2.1: the cell and the biological support that step S1 is obtained are by 106~107A cell/g biological support ratio
Mixing；
S2.2: configuring induced medium, add induced medium described in 10ml in the mixed system obtained to step S2.1,
Gently oscillation mixes 1min；The induced medium is β-phosphoglycerol of Indomethacin and 30mg/L added with 30mg/L
The DMEM-F12 culture solution of sodium；
S2.3: the mixed system that step S2.2 is obtained is added in microgravity rotating generator, stands 30min, rotation
30min is stood after 10min again, is repeated 3 times, rotating and culturing is started 1~2 week with the revolving speed of 15r/min, emptying in every 2 days is rotating
The bubble generated in incubation, every 3~4 days one subcultures of replacement.
Reference examples 2
A method of the adipose tissue for tissue transplantation, including each step described in embodiment 7 being prepared, wherein step S3
The method of middle addition cell growth helping matter is as follows:
Cell growth helping matter is added in bovine serum albumin(BSA) in 1% ratio and is uniformly mixed, 2mL is taken to be added to
In the culture that step S2 is obtained, gently oscillation mixes 3min.
Experimental example 1
At Adipose Differentiation performance test
Respectively in the method that embodiment 4,6,8 provides as experimental group 1~3, make respectively in the method that reference examples 1~2 provide
For control group 1~2, adipose tissue is prepared respectively using the mescenchymal stem cell of same source, is dyed after the completion of preparation with oil red
Identification fat drop is formed.
After rotating and culturing 3 days, cellular morphology i.e. change, gradually tapered up and shortened by spindle, 90% or more cell at
For cube or polygonal；There is small fat drop to occur after rotating and culturing 1 week, under mirror in visible cell, and fat drop with
The extension of incubation time is gradually expanded, merges；After cultivating 2 weeks, it is seen that merge pockets of fat drop full of entire cell.Oil red dye
The fat generated in color visible cell dyes red by specificity.Show effectively promote using preparation method provided by the invention
Into breaking up at rouge for mescenchymal stem cell.
Experimental example 2
Adipose tissue transplantation experiment
Respectively in the method that embodiment 5,7,9 provides as experimental group 1~3, make respectively in the method that reference examples 1~2 provide
For control group 1~2, adipose tissue is prepared respectively using the mescenchymal stem cell of same source, and distinguishes injection transplantation to rat
Below nipple, injection planes enter upper right side mammary gland as sky between mammary gland and chest muscle, while using ADSCs-COL gel injection
White control, every group injection 10.Periodic observation rat mammary gland skin conditions after injection.After 8 weeks, rat is anaesthetized and dissected, is observed
Whether there is or not cambiums to be formed, measurement cambium weight in wet base, while carrying out oil red dyeing to histotomy.
After injection, it is seen that the local eminence of mammary region.Have no that the obvious redness of skin or sense occurs in rat mammary gland during observation
The performance of the inflammation such as dye.After dissection, there is fat-like new life group between experimental group 1~3 and the visible mammary gland of control group 1~2 and chest muscle
It knits to be formed, adipose tissue is averaged weight in wet base as (138 ± 23) mg at experimental group injection, and adipose tissue is average at 1~2 injection of control group
Weight in wet base is (94 ± 17)；Blank control has no that obvious cambium is formed according to group, and the adipose tissue size weight in wet base that is averaged is at injection
(19±7)mg.The adipose tissue weight in wet base that is averaged is significantly higher than other groups at experimental group injection.Oil red dyes generation in visible cell
Fat dyes red by specificity, illustrates cambium for mature adipose tissue.The above result shows that using provided by the invention
The adipose tissue that preparation method obtains can quickly form new tissue after being implanted into vivo, promote the tissue of transplant recipient more
New and wound healing.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (9)
1. a kind of adipose tissue for tissue transplantation, which is characterized in that grown including biological support, fat cell and cell
Promote substance, the fat cell is inoculated into culture in the biological support by seed cell and obtains, between the seed cell is
Mesenchymal stem cells；
The biological support includes Type I collagen, chondroitin sulfate, chitosan, aminoglucan, polylactic acid, polyorthoester, poly- ε-
At least one of caprolactone and poly lactide-glycolide acid；
The cell growth helping matter include Butyrospermum parkii fruit rouge, asiaticosid, hydrolysis of pea albumen, creatine, allantoin with
And at least one of trehalose.
2. being used for the adipose tissue of tissue transplantation as described in claim 1, which is characterized in that connect in every gram of biological support
The number of the kind seed cell is 106~107It is a.
3. being used for the adipose tissue of tissue transplantation as described in claim 1, which is characterized in that the biological support includes weight
Portion rate is the polyorthoester and poly lactide-glycolide acid of 1:1~4.
4. being used for the adipose tissue of tissue transplantation as described in claim 1, which is characterized in that the cell growth helping matter
It is asiaticosid, creatine and the allantoin of 5:2~4:1~3 including weight fraction ratio.
5. a kind of method for preparing adipose tissue described in any one of Claims 1 to 4, which comprises the steps of:
S1: extraction mescenchymal stem cell, originally culture 2~3 days, and obtained cell is carried out secondary culture 1~2 week；
S2: the cell that step S1 is obtained is mixed in proportion with biological support, and addition first passes through the Fiber differentiation of ultrasonic treatment in advance
Base, gently oscillation mixes, and is placed in microgravity rotating generator and cultivates 1~2 week；
S3: to the obtained culture low-speed centrifugal of step S2 and discarding supernatant, and adds cell growth helping matter to get described in
Adipose tissue.
6. preparation method as claimed in claim 5, which is characterized in that the step S1 includes the following steps:
S1.1: extracting mescenchymal stem cell, wash 2 times with 30~45min of collagenase digesting, low-speed centrifugal and with PBS buffer solution,
By obtained cell inoculation into 10mL primary culture medium, it is placed in 5%CO2, cultivated in 37 DEG C of incubator；
S1.2: when cell fusion degree reaches 80~90%, adherent cell collecting presses 1000~6000 cell/cm after digestion2
Inoculum concentration be inoculated into 10mL secondary culture base, be placed in 5%CO2, cultivated in 37 DEG C of incubator, change liquid one within every 3 days
It is secondary, it cultivates until when cell fusion degree reaches 80~90%, is centrifuged, discards culture solution, and wash 2~3 with PBS buffer solution
It is secondary.
7. preparation method as claimed in claim 6, which is characterized in that the primary culture medium be added with volume fraction 5~
The DMEM-F12 culture solution of 15% fetal calf serum；
The secondary culture base is the DMEM-F12 training of the dense element of mandarin orange of growth factor and 5mg/L added with 50~100mg/L
Nutrient solution, the growth factor include the EGE and bFGF that weight fraction ratio is 1:1~1.5.
8. preparation method as claimed in claim 5, which is characterized in that the step S2 includes the following steps:
S2.1: the cell and the biological support that step S1 is obtained are by 106~107The mixing of a cell/g biological support ratio；
S2.2: configuration induced medium, and 5~10min of sonic oscillation under the conditions of 25 DEG C, 40~100Hz are stood after ultrasonic
It adds in the mixed system that induced medium described in 5~10mL is obtained to step S2.1, gently oscillation mixes 1~3min；Institute
Stating induced medium is Indomethacin added with 10~20mg/L, the vitamin C of 20~50mg/L and 10~20mg/L
The DMEM-F12 culture solution of sodium β-glycerophosphate；
S2.3: the mixed system that step S2.2 is obtained is added in microgravity rotating generator, 30~45min of standing, and rotation 5~
30~45min is stood after 10min again, repeats 3~4 times, rotating and culturing is started 1~2 week with the revolving speed of 15r/min, is emptied within every 2 days
The bubble generated during rotating and culturing, every 3~4 days one subcultures of replacement.
9. preparation method as claimed in claim 5, which is characterized in that in the step S3, add the cell growth and promote
The method of substance is as follows:
The cell growth helping matter is added in plant source recombination human serum albumin in 2~8% ratio and is mixed
Even, 5~10min of sonic oscillation under the conditions of 25 DEG C, 40~100Hz adds 2~5mL to step S2 after ultrasonic immediately and obtains
To culture in, gently oscillation mix 1~3min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592010A (en) * | 2019-10-14 | 2019-12-20 | 杨姣姣 | Application of asiatic acid in promoting in-vitro proliferation of human adipose tissue-derived mesenchymal stem cells and inducing chondrogenic differentiation of human adipose tissue-derived mesenchymal stem cells |
| CN110592009A (en) * | 2019-10-14 | 2019-12-20 | 杨姣姣 | Application of asiatic acid derivatives in promoting in-vitro proliferation and maintaining dryness of human adipose mesenchymal stem cells |
| CN114958731A (en) * | 2022-05-25 | 2022-08-30 | 大连医科大学 | Nano-pore silk fibroin scaffold-based engineered adipose tissue model, and preparation method and application thereof |
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2017
- 2017-11-30 CN CN201711241869.5A patent/CN109847096A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592010A (en) * | 2019-10-14 | 2019-12-20 | 杨姣姣 | Application of asiatic acid in promoting in-vitro proliferation of human adipose tissue-derived mesenchymal stem cells and inducing chondrogenic differentiation of human adipose tissue-derived mesenchymal stem cells |
| CN110592009A (en) * | 2019-10-14 | 2019-12-20 | 杨姣姣 | Application of asiatic acid derivatives in promoting in-vitro proliferation and maintaining dryness of human adipose mesenchymal stem cells |
| CN110592009B (en) * | 2019-10-14 | 2021-09-17 | 杨姣姣 | Application of asiatic acid derivatives in promoting in-vitro proliferation and maintaining dryness of human adipose mesenchymal stem cells |
| CN114958731A (en) * | 2022-05-25 | 2022-08-30 | 大连医科大学 | Nano-pore silk fibroin scaffold-based engineered adipose tissue model, and preparation method and application thereof |
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