CN101380333A - Method for extracting cellular fluid from human homogeneous variant cell - Google Patents
Method for extracting cellular fluid from human homogeneous variant cell Download PDFInfo
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- CN101380333A CN101380333A CNA200810211796XA CN200810211796A CN101380333A CN 101380333 A CN101380333 A CN 101380333A CN A200810211796X A CNA200810211796X A CN A200810211796XA CN 200810211796 A CN200810211796 A CN 200810211796A CN 101380333 A CN101380333 A CN 101380333A
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Abstract
The invention discloses a method for extracting cell sap from human allogeneic cells. The human allogeneic tissue cells are selected through a strict process and cultured in vitro cell, thereby becoming the cells containing a plurality of biological active ingredients. The cell sap is separated by the ultra-low-temperature extraction technology, the cells in the cell sap are treated by the physical wall-breaking, cytoplasm of the cells and nucleic acids, a plurality of short peptides, peptide cytokines, a plurality of amino acids, trace elements and the like in cell nucleus are released into the cell sap, thereby allowing the cell sap to be rich in the nucleic acids, the amino acids, natural immune proteins, cell growth factor groups, a plurality of collagen components and the like. The cell sap can be developed to a preparation with the cell biological active function, and the preparation can improve the cellular metabolism function of human body, enhance the immunity of the human body, improve the skin elasticity and the luster and the like after being acted on the human body. The cell sap has no toxicity or side effects through various safety detections.
Description
Technical field
The present invention relates to extract the preparation method of Cell sap, belong to molecular biology, cytobiology field from human homogeneous variant cell.
Background technology
Human body is made up of each system, and system is made up of various tissues, organizes then and is made up of various cells.Each cell is at every moment all carrying out metabolism in the lived body, and keep normal self-steady-state adjustment function just can keep normal in the various virulence factors that come from the outside of environment and opposing invasion and attack and do not produce disease.And cell carries out metabolism and keep self-stable state need consume various energy substances.As: nucleic acid, aminoacid, trace element, enzyme, immune protein, various cell growth factor, many types of collagen become to grade.
We choose the human homogeneous variant histiocyte by the flow process of strictness, choose the human homogeneous variant histiocyte by the flow process of strictness, cultivate through cell in vitro, become the cell that contains the various biological active component.Adopt the ultralow temperature abstraction technique to isolate Cell sap, after cell in the pair cell liquid carries out the physical wall breaking processing, the endochylema of cell and the nucleic acid in the nucleus, multiple small peptide, polypeptide cytokines, several amino acids, trace element etc. are discharged in the Cell sap, make and be rich in nucleic acid, aminoacid, natural immunity albumen, cell growth factor subgroup, many types of collagen in the Cell sap and become to grade.It can be developed to preparation, can improve body cell metabolic function, enhancing human body immunity power after acting on human body, and can improve effects such as skin elasticity and gloss with activity of cell biology function.Do not see toxic and side effects by every safety detection.
The base substance of synthetic proteins such as nucleic acid, aminoacid and trace element can improve the cell quality, and enzyme system is kept as normal catalysis; Body self and the damage of nature to body cell can be resisted and repair to natural immunity albumen; Cell growth factor subgroup (comprising transforming growth factor-beta, epithelical cell growth factor, vascular endothelial cell growth factor, fibroblast growth factor, stem cell factor etc.) with stimulating cellular growth effect is to the propagation of body cell (containing stem cell) and differentiation and metabolism, raising regenerative cell proportion, substitute to upgrade and originally have pivotal role because of factors such as aging or pathologic cause histiocytic decline and wear out etc.Because the metabolism of body cell is regulated and control to optimum state by the cytokine group, body cell keeps the ability of moisture also to strengthen greatly.Many types of collagen composition can promote that fibroblast propagation, fibroblast synthesize and the more collagen protein of secretion in the skin corium, repairs aging impaired collagen fiber and elastic fiber, and reduction skin elasticity is evenly compacted skin.Under cytokine group regulation and control, fibroblastic quantity increases greatly, and then fibroblast secretes continually from the body collagen protein.This synthesizing with excretory under corium from the body fibroblast can merge with body tissue better from the body collagen protein, make collagen protein support skin again, skin corium thickness and density increase, and then smooth away wrinkles, and make aged skin recover elasticity and gloss again.
Summary of the invention
The objective of the invention is to overcome the deficiency that prior art exists, a kind of preparation method of extracting Cell sap from human homogeneous variant cell is provided.
Preparation method from human homogeneous variant cell extraction Cell sap of the present invention comprises the steps:
(1) chooses healthy puerpera's fresh human placenta, umbilical cord and other allohisto compatibilities;
Tissues such as all Placenta Hominiss of suffering from hepatitis, malignant tumor, immunodeficiency, acquired immune deficiency syndrome (AIDS), various infectious disease, artificial abortion, stillborn fetus, various teratogenesis and umbilical cord must not use; Naked eyes detection outward appearance is undesired or have tissues such as rancid rotten Placenta Hominis, umbilical cord to use.
After Placenta Hominis is given birth to, put into sterilization container (food grade plastic bag) by the midwife with the sterile working immediately, and cold preservation immediately (below 10 ℃), in collecting transportation, also answer cold preservation.The placentology of collection resists-detection such as HBs, anti--HBc by the test kit of the national drug administrative authority approval of drawing.
The negative Placenta Hominis of HBV mark carries out anti-HCV, anti-HIV-1/HIV-2 and syphilis again and detects, and negative patient is qualified Placenta Hominis.
Detect qualified Placenta Hominis Ying Yu-20 ℃ frozen, and in Placenta Hominis was given birth to back 6 months, put into production.
(2) raw material is shredded be positioned in the container, spend pyrogen water and will shred tissue and clean, drain;
(3) add water for injection, open refiner with 8000~10000 rev/mins rotating speed homogenate;
(4) the homogenate branch is installed in the blood bag, seals, packing is obtained the blood bag after balance, place in the centrifuge 2500~3000 rev/mins centrifugal, get supernatant;
(5) with the hollow fiber column ultrafilter of supernatant through 50kD, filter liquor is gathered in the crops filtrate with the hollow fiber column ultrafiltration once more of 50kD, is stock solution;
(6) degerming, inactivation of viruses.
Compared with prior art, the present invention has following beneficial effect:
The present invention chooses the human homogeneous variant histiocyte by the flow process of strictness, cultivates through cell in vitro, becomes the cell that contains the various biological active component.Adopt the ultralow temperature abstraction technique to isolate Cell sap, after cell in the pair cell liquid carries out the physical wall breaking processing, the endochylema of cell and the nucleic acid in the nucleus, multiple small peptide, polypeptide cytokines, several amino acids, trace element etc. are discharged in the Cell sap, make and be rich in nucleic acid, aminoacid, natural immunity albumen, cell growth factor subgroup, many types of collagen in the Cell sap and become to grade.It can be developed to preparation, can improve body cell metabolic function, enhancing human body immunity power after acting on human body, and can improve effects such as skin elasticity and gloss with activity of cell biology function.Do not see toxic and side effects by every safety detection.
The specific embodiment
Embodiment 1
1. be engaged in the cell extract producers: should possess medical science or biology knowledge, and remote holder on duty is crossed strict training on operation.
2. production environment: separation chamber's cleanliness factor requires to be whole ten thousand grades, local laminar flow.
3. production equipment: high-speed homogenization machine, centrifuge, water bath, drying baker, capsuling machine (placing in the workshop), super-clean bench, blood separation folder, high-frequency electrical coupling sealing machine, clarity monitor station, electronic scale, PH meter, refrigerator, refrigerator-freezer, vacuum pump, superclean bench.
4. production apparatus: graduated cylinder, beaker, tweezers are (big, in, small size), two on shears, mosquito forceps, rustless steel pallet, micropipettor (1000ul, 100ul, 10ul), 30KD ultrafiltration pipe, alcohol burner, seal film, lighter, cillin bottle, bottle stopper, bottle cap, ethanol watering can 500ml, waste liquid cylinder, general utility balance.
5. easily-consumed products: 75% ethanol, pin type filter, asepticly secondaryly press bottle,suction, syringe (50ml, 20ml), deionized water, water for injection, micropipettor suction nozzle (1000ul, 200ul, 10ul), the aseptic apyrogeneity centrifuge tube of 45ml, 200ml blood bag, hospital gauze, tachypleus amebocyte lysate and detect water, virus detection reagent (HBs, anti-HCV, anti-HIV 1/2, syphilis detectable), cotton swab, 84 disinfectant solution, normal saline, aseptic apyrogeneity Stainless steel basin.Production meets drinking water standard with the source; Purified water and water for injection meet existing " Chinese pharmacopoeia standard.
6. raw-material purchase, transportation, reception, storage and quality control:
Meet the existing " requirement of Chinese pharmacopoeia or " the main raw and auxiliary material quality control standard of Chinese biological goods ".
6.1 choose healthy puerpera's fresh human placenta, umbilical cord and other allohisto compatibilities.Tissues such as all Placenta Hominiss of suffering from hepatitis, malignant tumor, immunodeficiency, acquired immune deficiency syndrome (AIDS), various infectious disease, artificial abortion, stillborn fetus, various teratogenesis and umbilical cord must not use; Naked eyes detection outward appearance is undesired or have tissues such as rancid rotten Placenta Hominis, umbilical cord to use.
After 6.2 Placenta Hominis is given birth to, put into sterilization container (food grade plastic bag) by the midwife with the sterile working immediately, and cold preservation immediately (below 10 ℃), in collecting transportation, also answer cold preservation.The placentology of collection resists-detection such as HBs, anti--HBc by the test kit of the national drug administrative authority approval of drawing.
6.3 the negative Placenta Hominis of HBV mark carries out anti-HCV, anti-HIV-1/HIV-2 and syphilis again and detects, negative patient is qualified Placenta Hominis.
6.4 detect qualified Placenta Hominis Ying Yu-20 ℃ frozen, and in Placenta Hominis was given birth to back 6 months, put into production.
7. preparation flow
Implement by China's " Good Manufacturing Practice and Quality Control of Drug " requirement.
7.1 production equipment is positioned over the appropriate location in workshop, check the ruuning situation of all production equipments, fill in report;
7.2 the cleaning of apparatus and equipment and apyrogeneity are handled:
Seal with masking foil and be positioned in the drying baker 250 ℃ of heating 1 hour 7.3 1 in 2000ml beaker, 500m14 are cleaned the back by the cleaning operation rules, take out after cooling and place in the super-clean bench ultraviolet disinfection standby.
High-speed homogenization machine rotor, shears with 1% 84 medicining liquid dippings after 1 hour, are cleaned 5 times and place in the super-clean bench ultraviolet disinfection standby with apirogen water.
7.4 the high-speed homogenization machine placed in the super-clean bench with 75% alcohol wipe sterilization sterilizes.
7.5 will be with 12 hours the ultrafiltration pipe (30kd) of 84 medicining liquid dippings of 200 times of dilutions with washed with de-ionized water 5 times, reuse water for injection cleans 5 times, place drain in the super-clean bench standby.
7.6 the processing of cillin bottle and bottle stopper, bottle cap is carried out according to corresponding cleaning operation rules are strict.
7.7 with micropipettor suction nozzle (1000ul, 200ul, 10ul), cotton swab, hospital gauze 1 bag, tweezers (big, in, little each two), two on shears, mosquito forceps 10 are 121 ℃ of, 3 autoclavings of rustless steel pallet, 30 minutes, dry for standby.
7.8 open workshop uviol lamp sterilization 1 hour.
Thawed 12 hours 7.9 raw material is positioned over 4 ℃, use the apyrogeneity masking foil to take by weighing the raw material of corresponding weight, shredding tissue to every block size with shears is 2 x, 2 x 2cm
3, will shred tissue and be positioned in the 500ml beaker.Spend pyrogen water and will shred tissue and clean 3 times, each water consumption is to shred 3 times of tissue, drains after washing.
7.10 add 1.5 times of waters for injection in the tissue after draining, open refiner with 8000~10000 rev/mins rotating speed homogenate 2 minutes to tissue.
7.11 homogenate is installed in the 200ml blood bag with 50ml syringe branch, and high-frequency electrical coupling sealing machine seals.
(above operation is sterile working in super-clean bench all)
7.12 the 200ml blood bag that packing is obtained after balance, place in the centrifuge 2500~3000 rev/mins centrifugal 30 minutes, and on the blood separation folder, operate and get supernatant; With the hollow fiber column ultrafilter of supernatant through 50kD, filter liquor is gathered in the crops filtrate with the hollow fiber column ultrafiltration once more of 50kD, is stock solution.
7.13 the ultrafiltration pipe is inserted in the 45ml centrifuge tube, in super-clean bench, the centrifuged supernatant of collecting is added in the ultrafiltration pipe, it is centrifugal that every pipe is no more than 5ml, and 3000 rev/mins are centrifugal 15 minutes;
Place in the 45ml apyrogeneity centrifuge tube 7.14 take out ultrafiltrate, through 0.22 μ m filtering with microporous membrane degerming 2 times;
(above Cell sap extraction work all places and carries out low-temperature operation on the ice bag)
7.15 carry out heat 10 hours inactivation of viruses 10 hours of 60 ℃ of water-baths.
8. cell in vitro is cultivated:
8.1 In vitro culture environment
Working environment requires cleaning, air drying and does not have flue dust.
8.1.1 aseptic: the strict sterile working that carries out is all wanted in thorough disinfection such as the air of culturing room, all operations, and various article should be sterilized before taking operating board, with spilling the smart surface of wiping, shines in ultraviolet with preceding; The operator washes one's hands, steeps hands, and alcohol hand is opened must iron on flame before the culture tube and burnt bottleneck and bottle, pipe tiltedly must be put during operation so that dust is fixed, and suction pipe injects liquid should be avoided contact with bottleneck, forbids during operation talking.
8.1.2 temperature: the optimum temperature of tissue culture is 35-37 ℃, departs from this temperature range, and the homergy of cell and growth will be affected, even cause death.Cultured cell is stronger than high temperature to cryogenic tolerance.Temperature increases 2-3 ℃ of pair cell and produces harmful effect, make it death in 24 hours, temperature is more than 43 ℃, and cell is killed mostly, and the influence of low temperature pair cell is less, when cell places 25-35 ℃, cell still can be survived and grow, but speed is slow, is placed on after 4 ℃ of a few hours, put 37 ℃ of cultivations again, cell still can continued growth.
8.1.3 gas: required gas mainly contains O
2And CO
2O
2Participate in the tricarboxylic acid cycle produce power and supply with cell growth, propagation and synthetic various compositions.CO
2Be products of cellular metabolism also be the required composition of cell.It main with keep medium pH value direct relation arranged.
8.1.4 pH value: the suitable pH value of most cells is 7.0-7.4, departs from this scope pair cell and can produce injurious effects.Buffer system in the culture fluid mainly is that buffered with bicarbonate is right.
8.1.5. cultivate the processing of vessel: most cells belongs to anchorage-dependent cell or culture.Glass drying oven must soak 1 to 7 day with cleaning solution, and the reuse distilled water is crossed and washed 2 times after the flushing with clean water 20 times, dries standby.Use after 2 hours with preceding 160 ℃ of high-temperature sterilizations.
8.2 passage cell leave standstill cultivation
The growing state that will examine cell every day determines whether will changing liquid or going down to posterity.With inverted microscope living cells form, endochylema and after birth are observed.Growth conditions good cell transparency is big, and the cell endoparticle is few, cannot see cavity, and after birth is clear, and refractivity is strong.Culture supernatant is Clear ﹠ Transparent, cannot see the cell and the fragment of suspension.During cell kakergasia, often occur cavity in the kytoplasm, fat drips and other granular substance, and the space is strengthened between the cell, and it is irregular that cellular morphology can become, even lose original characteristics.Have only cell in good condition just can be used for doing experiment.Generally at cell inoculation or after going down to posterity, every day or to multi-compartment 1-2 days answer observation of cell form, cell growth, culture fluid pH value and whether pollute etc., grasp the cell dynamic change at any time, change liquid or go down to posterity processing so that do.As unusual circumstance, in time take measures.Under the normal condition, it is pink that culture fluid is, and when cultivating with general incubator, with the prolongation of cell growth time, carbon dioxide build-up increases, because the existence of pH indicator is arranged in the culture medium, its color can reflect the growth conditions of cell indirectly.When being orange-yellow, the general growth conditions of cell is better; Being faint yellow then may be that incubation time is long, undernutrition, and dead cell is too much; Being aubergine then may be that cell growth state is bad or dead.
8.3 the preservation of cultured cell and recovery
8.3.1 influence the active factor of freeze-stored cell
8.3.1.1 cell cryopreservation protective agent: commonly used have dimethyl sulfoxide (DMSO) and glycerol, and concentration commonly used is 5%-10% (90% calf serum).DMSO is little than glycerol to the toxicity of diploid cell.
8.3.1.2 the freezing speed of cell suspension: freezingly when freezing slowly form, therefore induced damage resistive cell not in the extracellular.Most cells are reduced to-20 ℃ and are freezed with the speed of 1 ℃ of per minute decline.All can obtain promising result.
8.3.1.3 storage temperature: liquid nitrogen container, can reach-196 ℃, bottom line is all reduced in all physical chemistry activities this moment, preserves to guarantee cell long-period.
8.3.1.4 recovery: melt recovery rapidly and can prevent to damage cell.After freezing cell and from liquid nitrogen, taking out, should put into 37 ℃~43 ℃ water-bath immediately, melt in 30 seconds to one minute.
8.3.2. frozen and method for resuscitation:
8.3.2.1 cell is rare to 2~5 x 106/ml with cryopreserving liquid with trypsin+EDTA digestion back.
8.3.2.2 be sub-packed in the frozen pipe of 2ml, loading amount 1ml seals, and performs labelling, with which floor gauze ties.
8.3.2.3 frozen pipe should have a gradually process of cooling before entering liquid nitrogen, the speed cooling with 1 ℃/min generally hangs over liquid nitrogen after overhead 8 hours, drops into the liquid nitrogen container midium or long term and preserves.
8.3.2.4, frozen pipe is placed 37 ℃~43 ℃ water-baths for to make frozen cell recovery, fully shake it is melted rapidly, finish in 30 seconds to 1 minute.
8.3.2.5 the cell suspension low-speed centrifugal is removed the protection additive in the supernatant, adds the original growth-promoting media that uses, and puts 37 ℃ of cultivations.
8.4. reagent
8.4.1 be dissolved in No. three distilled water of new steaming 1640, to stir it is fully dissolved, filtration sterilization gets final product.
8.4.2 calf serum divides device-20 ℃ preservation standby with preceding 56 ℃ of deactivations in X30 minute (destruction complement).
8.4.3 the two anti-liquid of blue or green chain fully dissolve the penicillin sodium salt of 1,000,000 units and streptomycin sulfate with the tri-distilled water 100ml behind the high pressure 2 times, packing is frozen in-20 ℃, melt before using, add 1ml in every 100ml growth-promoting media, promptly use final concentration for containing P.S 100 μ g/ml.(annotate: P.S can not freeze thawing repeatedly, packing on a small quantity once uses up).
8.4.4 use NaHCO
3Liquid is adjusted pH value, and typical concentrations is 5~6%, and preparation is dissolved after-filtration degerming or autoclaving, 4 ℃ of preservations of packing with tri-distilled water.
8.4.5 trypsin: at pH 8.0, active force is the strongest during 37 ℃ of temperature.
8.4.6 EDTA liquid
8.4.7 cells frozen storing liquid: calf serum 90 ℅ mix with dimethyl sulfoxide (DMSO) 10 ℅;-20 ℃ of refrigerators are preserved standby.Or calf serum 30 ℅, RPMI-1640 60 ℅, DMSO10 ℅ mixes;-20 ℃ of refrigerators are preserved standby.
10. detect:
Undertaken by version rules appendix in 2000 " biological product chemistry and other calibration methods ".
10.1 aseptic experiment: undertaken by version rules general rule " biological product sterility test rules " A item in 2002;
10.2 endotoxin detects: by 2002 version rules general rule " biological product bacterial endotoxin test rules " carry out.Titre should be lower than 1:500; Intracellular toxin content should not be higher than 5EU/ml;
10.3 virus detects: every index is negative;
10.4 pyrogen test: by 2002 version rules general rule " biological product pyrogen test rules " carry out;
10.5 abnormal toxicity test: by 2002 version rules general rule " biological product abnormal toxicity test rules " carry out.
10.6 finger printing detects: 3 main bands should appear in the analysis of SDA-PAGE method; 66kd 23kd10kd
10.7 Protein Detection: should be about 1g/L;
10.8 pH: be 6.7~7.3;
10.9 content of peptides: measure with the BCA method, should be not less than 1.0mg/ml;
10.10 nucleic acid content: should be not less than 800 μ g/ml
10.11 color: should be colourless or little glistening yellow prescribed liquid, the foreign body that do not have, muddiness or precipitation.
11. in batches
By 2002 version rules general rule " biological product are rules in batches " carry out.
12. packing
By 2002 version rules general rule " biological product packing rules " carry out.
12.1 after qualified stock solution is adjusted to respective concentration after testing, uses the continuous filtration of 0.22um filter 2 times and filtrate placed the 45ml centrifuge tube;
12.2 use the 1000ul micropipettor, draw and be filled to cillin bottle filtrate branch, every bottle of 2.2ml adds a cover plug and plastic-aluminum;
(process of assembling must carry out in super-clean bench in above minute)
12.3 will add a cover the cillin bottle of plug and aluminium-plastic cap places on the capsuling machine and seals.
13. specification: every loading amount 2ml.Content of peptides should be no less than 2mg/ and prop up.
14. warehouse-in: through clarity detect qualified after, post label and be positioned under 2 ℃-8 ℃ the condition and preserve, carry out relative recording.
15. preservation, transportation and effect duration: 2.8 ℃ keep in Dark Place and transport, from Placenta Hominis feed intake from effect duration be 2 years.
Claims (1)
1. the preparation method from human homogeneous variant cell extraction Cell sap is characterized in that comprising the steps:
(1) chooses healthy puerpera's fresh human placenta, umbilical cord and other allohisto compatibilities;
(2) raw material is shredded be positioned in the container, spend pyrogen water and will shred tissue and clean, drain;
(3) add water for injection, open refiner with 8000~10000 rev/mins rotating speed homogenate;
(4) the homogenate branch is installed in the blood bag, seals, packing is obtained the blood bag after balance, place in the centrifuge 2500~3000 rev/mins centrifugal, get supernatant;
(5) with the hollow fiber column ultrafilter of supernatant through 50kD, filter liquor is gathered in the crops filtrate with the hollow fiber column ultrafiltration once more of 50kD, is stock solution;
(6) degerming, inactivation of viruses.
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102526109A (en) * | 2011-12-31 | 2012-07-04 | 上海善力健生物科技有限公司 | Method for extracting composite cell multiplication factors from placenta hominis |
CN102590417A (en) * | 2012-01-30 | 2012-07-18 | 云南民族大学 | Method for extracting inorganic negative ions from vegetable medicine at low temperature and ion chromatographic analysis method |
CN107550830A (en) * | 2017-08-31 | 2018-01-09 | 重庆金时代生物技术有限公司 | A kind of skin care solution and preparation method thereof |
CN113088549A (en) * | 2021-05-06 | 2021-07-09 | 内蒙古银宏干细胞生命科技投资有限公司 | Method for extracting antioxidant polypeptide from human placental blood |
-
2008
- 2008-09-25 CN CNA200810211796XA patent/CN101380333A/en active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102526109A (en) * | 2011-12-31 | 2012-07-04 | 上海善力健生物科技有限公司 | Method for extracting composite cell multiplication factors from placenta hominis |
CN102590417A (en) * | 2012-01-30 | 2012-07-18 | 云南民族大学 | Method for extracting inorganic negative ions from vegetable medicine at low temperature and ion chromatographic analysis method |
CN107550830A (en) * | 2017-08-31 | 2018-01-09 | 重庆金时代生物技术有限公司 | A kind of skin care solution and preparation method thereof |
CN113088549A (en) * | 2021-05-06 | 2021-07-09 | 内蒙古银宏干细胞生命科技投资有限公司 | Method for extracting antioxidant polypeptide from human placental blood |
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