CN1868420A - Human soft tissue filler for injection, and its prepn. method - Google Patents
Human soft tissue filler for injection, and its prepn. method Download PDFInfo
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Abstract
A filler of human soft tissue for injection is prepared through taking the skin tissue of patient itself, digestion, separating hypodermal fibrous stem cells, fibrous precursor cells and fibroblasts, external culture, and mixing the active components with the glucose injection or other injections.
Description
Technical field
The present invention relates to a kind of cell class tissue filling agent, be used for the filling and the reparation of human body soft tissue, comprise and remove wrinkle and repair depressed scar.The one-tenth fiber stem cell from body, one-tenth fiber precursor, fibroblast and collagen protein after In vitro culture, amplification in its tissue filling agent, have been comprised.Its preparation process has been used the serum-free culture material, finally is prepared into the injectable suspensions of tissue filling agent, is expelled in wrinkle, depressed scar, the traumatic dermis of skin and position such as lip, to reach filling, the purpose of reparation.
Background technology
Usually the method that is applied to the human body soft tissue filling can be divided into corium fills and subcutaneous filling, and these filleies were not autologous tissue's living materials in the past, but biosynthesis material or foreign preteins.Using packing material the earliest is at the 19th-century end, and it as filler, has occurred many complication with paraffin, and early stage, effect was also bad.The sixties have been used siloxane solution again as filler, have occurred part and systemic reaction too.Use extrinsic protein subsequently again as packing material, the collagen protein filler of cattle especially, this filler effect that can obtain medical treatment is is at once digested and assimilated but be organized in three to six months.The collagen protein of cattle preparation and utilize visible United States Patent (USP) 3,949,073 and 4,424,208 and 4,488,911.Its specification is 35mg/ml and 65mg/ml, and commodity are called ZydermI, ZydermII, ZydermIII, and after using Ryder, 90% patient has antibody to produce, and the patient of 1-3% has produced tangible anaphylaxis and can not be satisfactory; Thereby application glutaraldehyde cross-linking, filter with dusting cover then, produce the crosslinked bovine collagen albumen of a kind of Zyplast, thereby overcome reaction hypersensitive, its preparation process is seen United States Patent (USP) 4,582,640 and 4,642,117, but irregular problem has appearred again in its therapeutic effect, is because the tack of materials that obtains behind the glutaraldehyde cross-linking is too big.Showed to using the proteic experimenter's investigation of bovine collagen that the effect of a processing procedure generally can only continue the 4-6 month.
In order to eliminate the immunoreation of bovine collagen, there is the people from people's Placenta Hominis, to extract people's collagen, as the brand AUTOLOGEN that the U.S. once sold, see United States Patent (USP) 4,969,912 and 5,332,802, but the time that also can only keep some months.
The soft tissue filler that also occurred a kind of FIBREL of being called on the American market, it is the mixture of gelatin powder and glycine and patient's blood plasma, it is effective to the part patient, but is prone to problems such as injection site agglomerate and curative effect be not lasting.
Creotoxin (BOTOX) is a kind of widely used in recent years short-term injection reduce wrinkle liquid, obtains the approval of FDA in April, 2004, carries out clinical practice.Rely on it can make facial movement muscle anesthesia and reach therapeutic effect.Because said preparation is easy and simple to handle, take effect immediately and well-known, ossify but facial expression occurs after its treatment, curative effect is short, and also some the problem of facial paralysis occurs.
Artecoll (Artecoll) is synthetic granule and the proteic complex of bovine collagen, uses for many years in Canada, Mexico and Europe.Stimulate topical dermal to induce generation to reach permanently effective purpose from body collagen by this material, also occurred therapist on the curative effect and touched problems such as granular sensation is arranged, outstanding lump appears in the therapentic part of 16% therapist.
Owing to allosome material is injected the normal problems such as allergy, rejection and curative effect weak point that occur in back, American I solagen company begins to use a kind of Therapeutic Method of cultivating auto derma fibroblast reparation skin and soft tissue defects nineteen ninety-five and (sees United States Patent (USP) 5 in outpatient service, 591,444,5,665,372,5,660,850), its specific practice is, uses the skin histology of obtaining from body, and the culture fluid that contains Ox blood serum in external application is cultivated, be prepared at last in the Lin Gesi liquid and obtain from body fibroblast injection its no serious allergy, rejection.But U.S. FDA has proposed the requirement that limits the use of and forbid since 2000 for using animal serum injection preparation, that be used for the people, and the experimenter doubts the potential danger that has viruses such as bovine spongiform encephalopathy for Ox blood serum and has proposed query, thereby phase iii clinical trial has restarted in Isolagen company.Another weak point of this method is, Lin Gesi liquid be not easy to remain valid for a long time activity of composition needs very fast use after being mixed with injection, and is inconvenient in actual the use.
The Beijing Yiling Bioengineering Co., Ltd is since the injection (Chinese patent application 03155833.X number) of developing a kind of Wrinkle-and scar-removing that obtains in 2000, used autologous skin, through in-vitro separation, cultivation and amplification, be made in a kind of injection in the Glucose Liquid, treat cicatrix and dispel wrinkle.
With the culture fluid that partly contains serum the autologous skin sample being carried out tissue culture according to the effective ingredient in the injection of Chinese patent application 03155833.X obtains.Under analytical technology level at that time, effective ingredient wherein all is characterized as being fibroblast; But measure through existing analysis means, the effective ingredient after cultivation and the amplification not only contains fibroblast, also contains into the fiber stem cell, become fiber precursor and a spot of collagen protein.The weak point of this technology is, the collagen content that can bring into play the filling of human body soft tissue and repair function in the injection immediately is lower, causes being expelled to taking effect behind cicatrix and the wrinkle place slowlyer, generally takes 4-6 month; In addition, the patient also may have the doubt of safety aspect to using animal serum as the composition of culture fluid.
Recently, the applicant uses serum-free medium, from the autologous skin tissue, turned out and not only can comprise a large amount of one-tenth fiber stem cell, one-tenth fiber precursor and fibroblast, while but also contain abundant composite filler from the body collagen protein from body, at 1-3 month just obvious filling and repairing effect can appear, its instant effect, curative effect is lasting, natural safety.
Summary of the invention
An object of the present invention is to provide a kind of newly for the body human soft tissue filler for injection, it can be used to improve looks, improve the filling of skin elasticity and gloss and other position and type, and nature, curative effect are lasting.
Another object of the present invention provides this injection filler in beauty treatment, the filling that improves skin elasticity and gloss and other position and type and the application in the reparation.
Another object of the present invention provides the preparation method of this injection filler.
For achieving the above object, the present invention is by the following technical solutions:
Coming from therapist by human soft tissue filler for injection origin of the present invention separates through digestion from the skin of body, after the corium that obtains becomes fiber stem cell, one-tenth fiber precursor, fibroblast to carry out external omnidistance serum-free culture and amplification, be suitable for to become to be distributed into the glucose injection of concentration 5% or other injection and optional other again, effective ingredient becomes the fiber stem cell, becomes at least a and collagen protein in fiber precursor, the fibroblast to form by being selected from the corium of collecting after the cultivation; In every milliliter of filler, total number of effective cell composition is ten thousand of 1000-8000, and the content of collagen protein is 10-100mg/ml, preferred 20-60mg/ml.
A preferred version by tissue filling agent of the present invention, auto derma in the described filler becomes the fiber stem cell, becomes fiber precursor, fibroblast, collagen protein is to derive from a small amount of skin (skin of 1-30 square millimeter) of therapist from body, separate through digestion at cell culture chamber, the corium that obtains becomes the fiber stem cell, becomes fiber precursor, fibroblast, carries out In vitro culture and amplification and finally obtain in the serum-free medium that has added somatomedin and nutrient material; Adopted long In vitro culture and proliferation time, preferred 6-10 week.
A preferred version by tissue filling agent of the present invention, effective ingredient in the filler is become the fiber stem cell, is become fiber precursor, fibroblast and collagen protein and collagen protein to form jointly by said auto derma, and becoming the fiber stem cell, becoming the number proportion between fiber precursor, the fibroblast is 5-20 20-5 30-70; The part by weight 10-100mg/ml of collagen protein, preferred 20-60mg/ml injection filler.
Description of drawings
Accompanying drawing 1 is into fiber stem cell morphology photo under 100 times of optical microscopes;
Accompanying drawing 2 is into fiber precursor morphology photo under 100 times of optical microscopes;
Accompanying drawing 3 is fibroblast morphology photos under 100 times of optical microscopes.
The skin histology that the present invention gathers: comprise and behind ear, cut pouch, cut eyebrow, and other The skin that get at the position is the skin that comprises epidermis and corium.
Become fiber stem cell, Profibroblast, fibroblast and identifying with characteristic manner of collagen to be:
Become the fiber stem cell: (Huang Hui relies southwest, Wang Zhengguo, Wang Li to adopt the Brdu TPPA Beautiful, material is to the effect of Migration of Epidermal Stem Cells and expression of receptor in the wound healing; China's wound is assorted Will 2004; 20 (3) 142-145. utilize the nuclear label), cellular morphology can be attached referring to specification Fig. 1.
Profibroblast: under 100 times of light microscopes, do morphological observation, its canonical form Attitude is tiny long strand, and cellular morphology can be referring to Figure of description 2.
Fibroblast: do morphological observation under 100 times of light microscopes, its standard type is Be the prominent long rope shape cells that touch, cellular morphology can be referring to Figure of description 3 more.
Collagen: " adopting the collagen determination method ", Ministry of Health of the People's Republic of China's pharmacopeia The committee, the Pharmacopoeia of the People's Republic of China (two ones), Beijing, Chemical Industry Press, 2000.
Can be used in beauty treatment, improve the skin bullet according to human soft tissue filler for injection of the present invention The property and the filling of gloss and other position and type and repairing, comprise and processing because corium lacks The various symptoms of decreasing and causing, facial wrinkles for example, depressed scar, striae of pregnancy, the back of the hand wrinkle; Be used for rich lip, strengthen dermis thickness and improve dermal matrix etc.
The method for preparing human soft tissue filler for injection according to the present invention comprises autologous skin and gets Material, tissue digestion and cell separation, external free serum culture histocyte active ingredient and will have The glucose injection of effect composition and concentration 5% or other parenteral solution and choose wantonly other be suitable for into Be distributed into the several steps of injection fillers agent. Active ingredient is cultivated the rear corium one-tenth that obtains of collecting by being selected from At least a and collagen group in fiber stem cell, Profibroblast, the fibroblast Become; In every milliliter of filler, total number of effective cell composition is ten thousand of 1000-8000, glue The content of former albumen is 10-100mg/ml, preferred 20-60mg/ml.
A preferred version for preparing the method for human soft tissue filler for injection according to the present invention, also comprise somatomedin and active material in the culture fluid that uses in external omnidistance serum-free culture and amplification procedure, said somatomedin is at least a composition that is selected from epithelium growth factor, fibroblast growth factor; Said active material is at least a composition that is selected from 17-hydroxy-11-dehydrocorticosterone, hypophysis cerebri extracting solution, and these culture fluid and somatomedin and active material can derive from the commercially available prod an of company or a plurality of company (as the Cascad company of the U.S., Sigma company, Hyclne company etc.).The content of said somatomedin in culture fluid can be 1-10ng/ml, is preferably 2-5ng/ml; The content of said active material in culture fluid can be 0.1-1%, is preferably 0.2-0.5%.
A preferred version according to the inventive method comprises under the situation of somatomedin and active material in the culture fluid that uses in external omnidistance serum-free culture and amplification procedure, has also used long In vitro culture and proliferation time, is preferably 6-10 week.
According to a preferred version of the inventive method, the effective ingredient in the injection filler comprises said auto derma simultaneously and becomes the fiber stem cell, becomes fiber precursor, fibroblast and collagen protein; Becoming the fiber stem cell, becoming the number proportion between fiber precursor, the fibroblast is 5-20 20-50 30-70; The weight content scope 10-100mg/ml injection filler of collagen protein, preferred 20-60mg/ml injection filler.
According to another preferred version of the inventive method, take out frozen many years ago in liquid nitrogen autologous skin living cells (196 ℃), that after tissue digestion and cell separation, obtain, after thawing again, proceed to cultivate, amplification and other subsequent treatment.The benefit of doing like this is, can use preserve many years ago, than youth and the stronger autologous skin living cells of vitality, cultivate, increase and other subsequent treatment, skin is filled, is repaired and the effect of beauty treatment can be better.
Autologous skin is drawn materials
Disinfect the position of drawing materials in alcohol, carry out local surfaces anesthesia.Take off with cutisector or scalpel and surgical scissors epidermis and dermal tissue the 1-30 square millimeter, or excision eye pouch or cut the skin of eyebrow, put into tissue and preserve liquid, wound site to be made one to three pin sew up, adhesive bandage covers.
Tissue digestion and cell separation
The skin histology piece is positioned in the culture dish, adds pancreatin/EDTA liquid, DMEM+10%FBS liquid stops; Postdigestive piece of tissue is shredded and breaks up with suction pipe; Cell harvesting after dispelling to centrifuge tube, centrifugal 5 minutes, abandoning supernatant;
Cells in vitro is cultivated
Adopt tissue digestion culture method or tissue mass cell culture; Comprise the cultivation of primitive cell culture and passage cell.
Be by cell culture processes of the present invention and previous methods difference, adopt omnidistance serum-free culture mode first, in culture fluid, add somatomedin (as epithelium growth factor, fibroblast growth factor etc.) and active material (as 17-hydroxy-11-dehydrocorticosterone, hypophysis cerebri extracting solution etc.), cultivate 6-10 week, thereby promote the amplification of cell and the abundant secretion of collagen protein.Its cytoactive and form are normal, in the effective ingredient of collecting after the particularly this cultivation except that fibroblast from body being arranged, become the fiber stem cell, becoming the fiber precursor, also have relatively large collagen protein, it is 10-100mg/ml that its concentration can make the weight content scope of collagen protein in the injection filler after the preparation.
The composition of basic culture solution is well known to those of ordinary skill in the art, and particular content sees also " (cell experiment guide " (calendar year 2001, Science Press publishes, the chief editor: D.L. Spector, Huang Peitang translation); Adding above-mentioned somatomedin and/or active material on this basis again, promptly is the preferred culture fluid of the present invention.
Somatomedin can be epithelium growth factor (EGF), and its working concentration scope is 0.1-5ng/ml, and preferred concentration range for is 0.5-2ng/ml;
Active material can be hypophysis cerebri extracting solution (BPE), and its working concentration scope is 0.1-1%, and preferred concentration range for is 0.2-0.5%.
Incubation: will be 37 ℃ with above-mentioned serum-free medium in temperature from the isolated hypodermal cell of piece of tissue (contain into the fiber stem cell, become fiber precursor and fibroblast), 5% CO
2Cultivate in the cell culture incubator, changed in per three days and to state culture fluid once, cultivated altogether 42-70 days.
The filler preparation
Compound method according to human soft tissue filler for injection of the present invention comprises, with amplification in vitro as stated above and the glucose injection of the effective ingredient after cultivating and concentration 5% or other injection and other suitable composition of choosing wantonly be mixed with suspension, promptly be by injection filler of the present invention, effective ingredient becomes the fiber stem cell, becomes at least a and collagen protein in fiber precursor, the fibroblast to form by being selected from auto derma; In every milliliter of injection, the total number of effective cell composition is 1,000 ten thousand-8,000 ten thousand, and the content of collagen protein is the 10-100mg/ml injection.
Injection filler of the present invention itself has comprised a large amount of collagen protein, fills the effect of wrinkle and cicatrix in the very fast generation of injection part potential energy; Other effective ingredient that comprises in the injection filler also can continuously secrete a large amount of collagen protein, thereby rill and cicatrix length are put down, and is smoothing away wrinkles and the cicatrix while, can improve the environment of local skin again, plays the effect of young U.S. face.
Product applicable scope of the present invention is: fill and repair facial all wrinkles and depressed scar, as neck wrinkle, pregnant wrinkle, the back of the hand wrinkle, also be used for beauty treatment, strengthen the filling reparation at skin thickness, skin elasticity and other position.
Advantage of the present invention: employed effective ingredient derives from the patient from body, and no rejection can infection animal source property disease, have no side effect, the patient with after improve looks evident in efficacyly, look natural, instant effect, effect effect are lasting, can keep the several years.
The invention will be further described for the following specific embodiment.
The specific embodiment
Embodiment 1: the preparation of human soft tissue filler---be used to dispel evaluation of wrinkle treatment (20 example)
One. from the extraction of body healthy skin sample
Behind 70% alcohol disinfecting ear, use then 10% lignocaine and 100,000/ epinephrine carry out local surfaces anesthesia.With cutisector or scalpel and surgical scissors with the curer ear after 4 square millimeters epidermis and dermal tissue take off, put into tissue and preserve in the liquid (DMEM cell-preservation liquid, U.S. Hyclone company), wound site is made a pin sews up, adhesive bandage covers.
Two. the preparation of cell suspension (tissue digestion culture method)
1. tissue digestion (Forma, or Sanyo CO
2Incubator): the skin histology piece is positioned in the 35mm culture dish, and adding 1ml concentration is pancreatin/EDTA liquid of 0.05%, 37 ℃, and 10 minutes; DMEM+10%FBS liquid stops.
2. cell separation: postdigestive piece of tissue is shredded and breaks up with suction pipe.
3. centrifugal: the cell harvesting after dispelling to centrifuge tube, Backman company temperature control centrifuge, 25 ℃, 1000 rev/mins, centrifugal 5 minutes, abandoning supernatant.
4. primitive cell culture: in centrifuge tube, add 1ml hypodermal cell culture fluid (Cascade company, the trade mark is 106, wherein main component is a basic culture solution, the concentration of somatomedin EGF is 1ng/ml, the concentration of nutrient material BPE is
0.2%, the U.S.) to blow outstandingly, the 35mm culture dish is gone in inoculation; CO
2Incubator, 37 ℃, 5%CO
2, cultivate;
5. passage cell is cultivated: changed every 2～3 days and state culture fluid 1 time, go down to posterity after 50-70% compiles when primary cell reaches, with trypsinization, centrifugal, abandoning supernatant, cultivate by 1: 3 amplification times.
6. preparation injection: be collected in the effective ingredient in subculture in vitro separately to the culture in 8 weeks; To concentration is to add the effective ingredient of collecting in 5% the glucose injection, makes every milliliter of suspension contain ten thousand effective cell compositions of about 2000-6000, is mixed with the 1-4ml suspension.
Three. product detects
1. virus detects: after skin histology was cultivated for 6 weeks, get the culture fluid that 1ml contains cell and carry out the check of HIV and hepatitis virus, determine that this is virus-free cell.
2. immunoreation test: the 6th week was carried out skin test (with penicillin skin test method), no immunoreation with the 0.1ml cell suspending liquid to the operation receiveing person.
3. finished product detection
1) outward appearance: this product is the pale suspension.
Effective ingredient classification calibrating in this product:
Become the fiber stem cell: adopt the Brdu TPPA to show, the ratio that becomes the number of fiber stem cell to account for three kinds of total numbers of cell in this filler is 10-20%.
Become the fiber precursor: adopt the morphocytology method to observe, the ratio that becomes fiber precursor number to account for three kinds of total numbers of cell in this filler is 30-40%.
Fibroblast: adopt the morphocytology method to observe, to account for the ratio of three kinds of total numbers of cell be 50-70% to the fibroblast number in this filler.
Collagen protein: adopt the collagen protein assay method, measure that collagen protein composition weight content is 20-60mg/ml in this filler.
2) sterility test: undertaken by " Chinese biological goods rules " (2000 editions) general rule " biological product sterility test rules " A/B item, the result meets aseptic requirement.
The storage of product and transportation are in 4 ℃ special preservation case, and this case meets cool place, drying, cleans, avoids the sunlight direct projection, the condition of non-corrosiveness gas, no weight.
Four. treatment
Be used for the forehead wrinkle, canthus fishtail line, neck wrinkle, all wrinkle and tiny wrinkles facial and other body part such as striae gravidarum; Treat 25 examples.
1. check before the therapist art that every index is all normal, comprising: hematuria is routine, electrocardiogram, hepatic and renal function, HIV, HbsAg just.
With the injection site with 70% alcohol disinfecting, the lignocaine anesthesia of local injection 1%.
3. standby in the syringe with 1.5ml cell suspension suction 3ml.
4. get No. 4.5 long syringe needles of 2.2cm, adopt multiple spot inclined-plane (20 °～45 ° of the angles of syringe needle and skin), when injecting,, it is turned white, make the injection site leave the disperse space during injection injection site epidermis tension injection cell in the injection site.Inject the back and used ice bag external application 2 hours.
The course of treatment: the injection site is injected 2 times altogether, at interval 2 weeks.
Five, clinical follow, nurse:
1. after the operation, apply injection place 2 hours with ice bag.
2. observe the situation of part and whole body: all are normal.
3. secondary oral vitamin C every day (each 200mg, every day is 400mg altogether) took 6 months.
4. postoperative was prevented tanning by the sun and the careful zest cosmetics of using in 3 days.
Six, effect is described:
Wrinkle disappears or obviously shoals in that injection is basic after back three months, and is respond well.
Embodiment 2: the preparation of human soft tissue filler---dispel depressed scar (20 example)
One. from the extraction of body healthy skin sample
Behind 70% alcohol disinfecting ear, use then 10% lignocaine and 100,000/ epinephrine carry out local surfaces anesthesia.With cutisector or scalpel and surgical scissors with the curer ear after 4 square millimeters epidermis and dermal tissue take off, put into tissue and preserve in the liquid (DMEM cell-preservation liquid, U.S. Hyclone company), wound site is made a pin sews up, adhesive bandage covers.
Two. the preparation of cell suspension
1. tissue mass cell culture: piece of tissue is chopped into 1 * 1mm
2, be tiled in the 35mm culture dish, add the above-mentioned hypodermal cell culture fluid of 2ml (Cascade company, the trade mark is 106, and wherein main component is a basic culture solution, and the concentration of somatomedin EGF is 1ng/ml, the concentration of nutrient material BPE is 0.2%, the U.S.) carry out primitive cell culture.
2. passage cell is cultivated: changed every 2～3 days and state culture fluid 1 time, go down to posterity after 50-70% compiles when primary cell reaches, with trypsinization, centrifugal, abandoning supernatant, cultivate by 1: 3 amplification times.
3. preparation injection: collect the effective ingredient in the culture in 8 weeks of subculture in vitro separately to the; To concentration is to add the effective ingredient of collecting in 5% the glucose injection, makes every milliliter of suspension contain ten thousand effective cell compositions of about 3000-7000, is mixed with the 1-4ml suspension.
Three. product detects
1. virus detects: after skin histology was cultivated for 8 weeks, get the culture fluid that 1ml contains cell and carry out the check of HIV and hepatitis virus, determine that this is virus-free cell.
2. immunoreation test: the 8th week was carried out skin test (with penicillin skin test method), no immunoreation with the 0.1ml cell suspending liquid to the operation receiveing person.
3. finished product detection
1) outward appearance: this product is the pale suspension.
Effective ingredient classification calibrating in this product:
Become the fiber stem cell: adopt the Brdu TPPA to show, the ratio that becomes fiber stem cell number to account for three kinds of total numbers of cell in this filler is 15-20%.
Become the fiber precursor: adopt the morphocytology method to observe, the ratio that becomes fiber precursor number to account for three kinds of total numbers of cell in this filler is 20-50%.
Fibroblast: adopt the morphocytology method to observe, to account for the ratio of three kinds of total numbers of cell be 30-50% to the fibroblast number in this filler.
Collagen protein: adopt the collagen protein assay method, measure that collagen protein composition weight content is 30-80mg/ml in this filler.
2) sterility test: undertaken by " Chinese biological goods rules " (2000 editions) general rule " biological product sterility test rules " A/B item, the result meets aseptic requirement.
The storage of product and transportation are in 4 ℃ special preservation case, and this case meets cool place, drying, cleans, avoids the sunlight direct projection, the condition of non-corrosiveness gas, no weight.
Four. treatment
Facial acne, the variola cicatrix, traumatic, depressed scar is treated 20 examples.
1. check before the therapist art that every index is all normal, comprising: hematuria is routine, electrocardiogram, hepatic and renal function, HIV, HbsAg just.
With the injection site with 70% alcohol disinfecting, the lignocaine anesthesia of local injection 1%.
3. standby in the syringe with 1.5ml cell suspension suction 3ml.
4. get No. 4.5 long syringe needles of 2.2cm, adopt multiple spot inclined-plane (20 °～45 ° of the angles of syringe needle and skin), at injection site injection cell suspension.With the tension of injection site epidermis, it is turned white during injection, make the injection site leave the disperse space during injection.Inject the back and used ice bag external application 2 hours.
The course of treatment: inject 2 times altogether at above-mentioned position, at interval 2 weeks.
Five, clinical follow, nurse:
With embodiment 1.
Six, effect is described:
Behind the treatment one and a half months, the cicatrix performance of its pitting is smooth, and with the surrounding skin basically identical, skin is glossy, and is respond well.
Embodiment 3: the preparation of human soft tissue filler---be used for the improvement and the beauty treatment (20 example) of dermal matrix
One. from the extraction of body healthy skin sample
Behind 70% alcohol disinfecting ear, use then 10% lignocaine and 100,000/ epinephrine carry out local surfaces anesthesia.With cutisector or scalpel and surgical scissors with the curer ear after 4 square millimeters epidermis and dermal tissue take off, put into tissue and preserve in the liquid (DMEM cell-preservation liquid, U.S. Hyclone company), wound site is made a pin sews up, adhesive bandage covers.
Two. the preparation of cell suspension
1. tissue mass cell culture: piece of tissue is chopped into 1 * 1mm
2, be tiled in the 35mm culture dish, and the above-mentioned hypodermal cell culture fluid of adding 2ml (Cascade company, the trade mark is 106, and wherein main component is a basic culture solution, and the concentration of somatomedin EGF is 1ng/ml, nutrient material
BPEConcentration be 0.2%, the U.S.) carry out primitive cell culture.
2. passage cell is cultivated: changed every 2～3 days and state culture fluid 1 time, go down to posterity after 50-70% compiles when primary cell reaches, with trypsinization, centrifugal, abandoning supernatant, cultivate by 1: 3 amplification times.
3. preparation injection: collect the effective ingredient in the culture in 8 weeks of subculture in vitro separately to the; To concentration is to add the effective ingredient of collecting in 5% the glucose injection, makes every milliliter of suspension contain ten thousand effective cell compositions of about 1500-5500, is mixed with the 1-4ml suspension.
Three. product detects
1. virus detects: after skin histology was cultivated for 8 weeks, get the culture fluid that 1ml contains cell and carry out the check of HIV and hepatitis virus, determine that this is virus-free cell.
2. immunoreation test: the 8th week was carried out skin test (with penicillin skin test method), no immunoreation with the 0.1ml cell suspending liquid to the operation receiveing person.
3. finished product detection
1) outward appearance: this product is the pale suspension.
Effective ingredient classification calibrating in this product:
Become the fiber stem cell: adopt the Brdu TPPA to show, the ratio that becomes fiber stem cell number to account for three kinds of total numbers of cell in this filler is 5-20%.
Become the fiber precursor: adopt the morphocytology method to observe, the ratio that becomes fiber precursor number to account for three kinds of total numbers of cell in this filler is 20-50%.
Fibroblast: adopt the morphocytology method to observe, to account for the ratio of three kinds of total numbers of cell be 30-70% to the fibroblast number in this filler.
Collagen protein: adopt the collagen protein assay method, measure that collagen protein composition weight content is 15-50mg/ml in this filler.
2) sterility test: undertaken by " Chinese biological goods rules " (2000 editions) general rule " biological product sterility test rules " A/B item, the result meets aseptic requirement.
The storage of product and transportation are in 4 ℃ special preservation case, and this case meets cool place, drying, cleans, avoids the sunlight direct projection, the condition of non-corrosiveness gas, no weight.
Four. treatment
Treatment skin out-of-flatness has the skin microgroove, and skin gloss dimness and be used for abundant lip is filled nasolabial fold etc., treats 20 examples.
1. check before the therapist art that every index is all normal, comprising: hematuria is routine, electrocardiogram, hepatic and renal function, HIV, HbsAg just.
With the injection site with 70% alcohol disinfecting, the lignocaine anesthesia of local injection 1%.
3. standby in the syringe with 1.5ml cell suspension suction 3ml.
4. get No. 4.5 long syringe needles of 2.2cm, adopt multiple spot inclined-plane (20 °～45 ° of the angles of syringe needle and skin), at injection site injection cell suspension.With the tension of injection site epidermis, it is turned white during injection, make the injection site leave the disperse space during injection.Inject the back and used ice bag external application 2 hours.
The course of treatment: inject 2 times altogether at above-mentioned position, at interval 2 weeks.
Five, clinical follow, nurse:
With embodiment 1.
Six, effect is described:
Treat after three months, skin is smooth substantially, and tiny stricture of vagina disappears, and it is bright and clean flexible that skin becomes; It is more plentiful that lip seems, nasolabial fold obviously shoals.
Human soft tissue filler for injection treatment case statistical report (case load 60)
1. include standard in: the age (comprised 60 years old) below 60 years old; The wrinkle person be forehead, near the eyes, perioral aging lines; The cicatrix person is acne, wound cicatrix (magnitude range no requirement (NR)), cutis laxa, gloss dimness etc.
2. exclusion standard: the age is in therapist more than 60 years old; Autoimmune disease is arranged; The chronic skin patient is arranged; The infection patient is arranged.
3, through to the analysis of cases of 60 examples, double injection 48 examples are arranged, continue 6 months observing times.The result is, begins in 3 weeks from accepting injection treatment, has 30 examples to demonstrate the obvious filling repairing effect, and therapist feels satisfied 60 examples (its satisfaction rate is 100%) that have; During by 3 months, the wrinkle after the treatment disappears substantially, and cicatrix obviously reduces, and the dermal matrix situation obviously improves.
Wherein there are several therapists to observe so far 2 years, the complete obiteration of its wrinkle, cicatrix, skin smooth and high resilience.
Comparative example: the preparation and the application of prior art Wrinkle-and scar-removing injection
According to the method for record in Chinese patent application 03155833.X number, prepared the Wrinkle-and scar-removing injection of prior art, and comparison test its effect, specific practice such as following.
One. from the extraction of body healthy cell
With behind 70% the alcohol disinfecting experimenter ear or arm inboard, use then 10% lignocaine and 100,000/ epinephrine carry out local surfaces anesthesia.With cutisector with ear after 4 square millimeters epidermis and dermal tissue take off, put into tissue and preserve in the liquid (DMEM and 10% hyclone, U.S. Hyclone company), wound site is made a pin sews up, adhesive bandage covers.
Two. the preparation of cell suspension
1. tissue digestion (Sanyo CO
2Incubator): the skin histology piece is positioned in the 35mm culture dish to add 2ml concentration be pancreatin/EDTA liquid of 0.05%, 37 ℃, 10 minutes; The DMEM+10%FBS culture fluid stops.
2. cell separation: postdigestive piece of tissue is shredded and breaks up with suction pipe.
3. centrifugal: the cell harvesting after dispelling to centrifuge tube, Backman company temperature control centrifuge, 25 ℃, 1000 rev/mins, centrifugal 5 minutes, abandoning supernatant.
4. primitive cell culture: add 1ml and contain serum true chrotoplast culture fluid (DMEM, D5546 add hyclone F2242, Sigma company, the U.S.) and blow outstandingly in centrifuge tube, 1 * 35mm culture dish is gone in inoculation; Sony CO
2Incubator, 37 ℃, 5%CO
2, cultivate.
5. passage cell is cultivated: changed liquid 1 time every 2～3 days, go down to posterity after 50-70% compiles cell when cell reaches.
6. preparation injection: be collected in the effective ingredient of subculture in vitro separately to the after 5 weeks, in 5% glucose injection, add the effective ingredient of collecting, be mixed with the 1-4ml suspension that every milliliter of suspension contains ten thousand effective cell compositions of 1000-2000.
Three. product detects
1. virus detects: after skin histology was cultivated for 5 weeks, get the check that culture fluid that 1ml contains cell send Beijing infectious hospital to carry out HIV and hepatitis virus, be virus-free cell.
2. immunoreation test: the 0.1ml cell suspending liquid is carried out skin test (with penicillin skin test method) to the operation receiveing person, no immunoreation around the.
3. finished product detection
1) outward appearance: this product is the pale suspension.
Effective ingredient classification calibrating in this product:
Become the fiber stem cell: adopt the Brdu TPPA to show, the ratio that becomes fiber stem cell number to account for three kinds of total numbers of cell in this filler is 5-15%.
Become the fiber precursor: adopt the morphocytology method to observe, the ratio that becomes fiber precursor number to account for three kinds of total numbers of cell in this filler is 30-50%.
Fibroblast: adopt the morphocytology method to observe, to account for the ratio of three kinds of total numbers of cell be 40-60% to the fibroblast number in this filler.
Collagen protein: adopt the collagen protein assay method, measure that collagen protein composition weight content is 2-5mg/ml in this filler.
2) sterility test: undertaken by " Chinese biological goods rules " (2000 editions) general rule " biological product sterility test rules " A/B item, the result meets aseptic requirement.
The storage of product and transportation are in 4 ℃ special preservation case, and this case meets cool place, drying, cleans, avoids the sunlight direct projection, the condition of non-corrosiveness gas, no weight.
Four. treatment
The treatment facial wrinkles, depressed scar, 28 examples.
1. inspection is all normal before patient's art, and comprising: hematuria is routine, electrocardiogram, hepatic and renal function, HIV, HbsAg just.
With the injection site with 70% alcohol disinfecting, epidermis is anaesthetized with 2.5% lignocaine.
3. standby in the syringe with 1.5ml cell suspension suction 3ml.
4. get No. 4.5 long syringe needles of 2.2cm, adopt multiple spot inclined-plane (20 °～45 ° of the angles of syringe needle and skin) cell suspension to be injected into the upper strata and the middle level of wrinkle and depressed scar skin corium, the injection site epidermis should be strained during injection, it is turned white, should make the injection site leave the disperse space during injection.Inject the back and used ice bag external application 2 hours.
Postoperative does not have other part and general reaction.
The course of treatment: inject 2 times, per injection is 2 weeks at interval.
Five, clinical follow, nurse:
1. after the operation, apply injection place 2 hours with ice bag.
2. observe the situation of part and whole body.
3. the secondary part was put on the skin and was contained ascorbic facial cream, about 6 months every day.
4. postoperative was prevented tanning by the sun and the careful zest cosmetics of using in 3 days.
Six, effect is described:
Treat after six months, canthus fishtail line disappears substantially, and the forehead wrinkle shoals and part disappears, and forehead skin is glossy, and elasticity strengthens.Therapist generally believes, needs a 6-9 month rear positive effect to occur, and onset time dislikes slow.
Claims (11)
1, a kind of human soft tissue filler for injection, it is characterized in that: origin comes from therapist and separates through digestion from the skin of body, after the corium that obtains becomes fiber stem cell, one-tenth fiber precursor, fibroblast to carry out external omnidistance serum-free culture and amplification, the glucose injection of the effective ingredient collected and concentration 5% or other injection and optional other are suitable for to become to be distributed into, and effective ingredient becomes the fiber stem cell, becomes at least a and collagen protein in fiber precursor, the fibroblast to form by being selected from the corium of collecting after the cultivation; In every milliliter of injection filler, total number of effective cell composition is ten thousand of 1000-8000, and the content of collagen protein is 10-100mg/ml injection filler.
2, human soft tissue filler for injection according to claim 1, it is characterized in that: the effective ingredient in the injection filler is become the fiber stem cell, is become fiber precursor, fibroblast and collagen protein to form jointly by said auto derma, and becoming the fiber stem cell, becoming the number proportion between fiber precursor, the fibroblast is 5-20: 20-50: 30-70; The content of collagen protein is 10-100mg/ml injection filler, preferred 20-60mg/ml injection filler.
3, human soft tissue filler for injection according to claim 2 is characterized in that: said effective ingredient is to obtain used the culture fluid that also comprises somatomedin and active material in external omnidistance serum-free culture and amplification procedure after.
4, human soft tissue filler for injection according to claim 3, it is characterized in that: said somatomedin is at least a composition that is selected from epithelium growth factor, fibroblast growth factor etc., its content range in culture fluid is 1-10ng/ml, is preferably 2-5ng/ml; Said active material is at least a composition that is selected from 17-hydroxy-11-dehydrocorticosterone, hypophysis cerebri extracting solution etc., and its content range in culture fluid is 0.1-1%, is preferably 0.2-0.5%.
5, according to the application of any described human soft tissue filler for injection among the claim 1-4, be used for the damaged and filling of the various symptoms that cause of corium, comprise the process facial wrinkle, the cervical region wrinkle, depressed scar, striae gravidarum, the back of the hand wrinkle, rich lip, and strengthen dermis thickness and improve dermal matrix.
6, a kind of method for preparing human soft tissue filler for injection, comprise that autologous skin is drawn materials, tissue digestion and cell separation, external serum-free culture histiocyte effective ingredient become to be distributed into the filler several steps with the glucose injection of effective ingredient and concentration 5% or other injection and optional other are suitable for, effective ingredient becomes the fiber stem cell, becomes at least a and collagen protein in fiber precursor, the fibroblast to form by being selected from the corium of collecting after the cultivation; In every milliliter of filler, total number of effective cell composition is ten thousand of 1000-8000, and the content of collagen protein is 10-100mg/ml injection filler.
7, method according to claim 6 is characterized in that, effective ingredient is become the fiber stem cell, become fiber precursor, fibroblast and collagen protein to form jointly by said auto derma; Becoming the fiber stem cell, becoming the number proportion between fiber precursor, the fibroblast is 5-20: 20-50: 30-70; The content 10-100mg/ml injection filler of collagen protein, preferred 20-60mg/ml injection filler.
8, method according to claim 7, it is characterized in that, also comprise somatomedin and active material in the culture fluid that uses in external omnidistance serum-free culture and amplification procedure, said somatomedin is at least a composition that is selected from epithelium growth factor, fibroblast growth factor; Said active material is at least a composition that is selected from 17-hydroxy-11-dehydrocorticosterone, hypophysis cerebri extracting solution.
9, method according to claim 8 is characterized in that, the content of said somatomedin is 0.1-5ng/ml in the culture fluid, is preferably 0.5-2ng/ml; The content of said active material is 0.1-1%, is preferably 0.2-0.5%.
10, according to any described method among the claim 6-9, it is characterized in that the In vitro culture time is 6-10 week.
11, method according to claim 10, it is characterized in that, take out frozen many years ago in liquid nitrogen autologous skin living cells (196 ℃), that after tissue digestion and cell separation, obtain, after thawing again, proceed to cultivate, amplification and other subsequent treatment.
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| CN100421731C (en) * | 2006-01-11 | 2008-10-01 | 黄靖西 | Injection liquid of human autogenous collagen living cell and preparation method thereof |
| CN101919380A (en) * | 2010-08-06 | 2010-12-22 | 青岛奥克生物开发有限公司 | Improved mesenchyme stem cell protection solution and application thereof |
| CN102205146A (en) * | 2011-04-12 | 2011-10-05 | 王影 | Stem cell repairing material as well as preparation method and application thereof |
| CN104984395A (en) * | 2015-04-28 | 2015-10-21 | 中国人民解放军第四军医大学 | Autologous-tissue-based activity extract for soft tissue filling and preparation method |
| CN109432499A (en) * | 2018-10-31 | 2019-03-08 | 白晋 | A kind of preparation method of the autologous collagen protein injection agent of combination secret reparation |
| CN114015648A (en) * | 2021-10-20 | 2022-02-08 | 成都拜美森生物科技有限公司 | High-performance adipose-derived mesenchymal stem cell solution and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100421731C (en) * | 2006-01-11 | 2008-10-01 | 黄靖西 | Injection liquid of human autogenous collagen living cell and preparation method thereof |
| CN101919380A (en) * | 2010-08-06 | 2010-12-22 | 青岛奥克生物开发有限公司 | Improved mesenchyme stem cell protection solution and application thereof |
| CN101919380B (en) * | 2010-08-06 | 2014-10-22 | 青岛奥克生物开发有限公司 | Improved mesenchyme stem cell protection solution and application thereof |
| CN102205146A (en) * | 2011-04-12 | 2011-10-05 | 王影 | Stem cell repairing material as well as preparation method and application thereof |
| CN104984395A (en) * | 2015-04-28 | 2015-10-21 | 中国人民解放军第四军医大学 | Autologous-tissue-based activity extract for soft tissue filling and preparation method |
| CN109432499A (en) * | 2018-10-31 | 2019-03-08 | 白晋 | A kind of preparation method of the autologous collagen protein injection agent of combination secret reparation |
| CN114015648A (en) * | 2021-10-20 | 2022-02-08 | 成都拜美森生物科技有限公司 | High-performance adipose-derived mesenchymal stem cell solution and preparation method and application thereof |
| CN114015648B (en) * | 2021-10-20 | 2024-02-20 | 成都拜美森生物科技有限公司 | High-performance adipose-derived mesenchymal stem cell solution and preparation method and application thereof |
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| CN100581502C (en) | 2010-01-20 |
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