CN105316309A - Digestive enzyme composition and preparation method of fibroblasts and feeder layer cells - Google Patents

Digestive enzyme composition and preparation method of fibroblasts and feeder layer cells Download PDF

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Publication number
CN105316309A
CN105316309A CN201510889246.3A CN201510889246A CN105316309A CN 105316309 A CN105316309 A CN 105316309A CN 201510889246 A CN201510889246 A CN 201510889246A CN 105316309 A CN105316309 A CN 105316309A
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embryo
preparation
cell
dispase
trypsinase
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陈海佳
葛啸虎
王一飞
罗二梅
张维敏
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Guangzhou Saliai StemCell Science and Technology Co Ltd
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Guangzhou Saliai StemCell Science and Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/6416—Metalloendopeptidases (3.4.24)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21004—Trypsin (3.4.21.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24004—Microbial metalloproteinases (3.4.24.4)

Abstract

The invention relates to the technical field of cell culture, and particularly relates to a digestive enzyme composition and a preparation method of fibroblasts and feeder layer cells. The digestive enzyme composition comprises trypsin and dispase. In the digestive enzyme composition disclosed by the invention, after the trypsin and the dispase are jointly used in a certain proportion, a good digestion effect can be achieved under the condition of greatly reducing the application amount of digestive enzymes, so that the yield of fibroblasts is higher.

Description

The preparation method of a kind of digestive enzyme compositions and inoblast and feeder layer cells
Technical field
The present invention relates to technical field of cell culture, particularly the preparation method of a kind of digestive enzyme compositions and inoblast and feeder layer cells.
Background technology
Embryonic stem cell (embryonicstemcell, ESCs, be called for short ES, EK or ESC cell) be the class cell separated in body early embryo (before gastrula stage) or original sexual gland, it has the characteristic of vitro culture infinite multiplication, self and Multidirectional Differentiation.No matter in vitro or internal milieu, ES cell can be induced to differentiate into the nearly all cell type of body.Embryonic stem cell has using value in the field such as organizational project and regenerative medicine.
But, be difficult to obtain due to it and relate to responsive ethics problem, making research once be absorbed in awkward condition.In August, 2006, Yamanaka research group of Japan imports 4 kinds of transcription factor genes (Oct4, Sox2, c-Myc and Klf4) in l cell, be successfully ESCs sample inductive pluripotent stem cells (inducedpluripotentstemcells, iPSCs) by its reprogrammed.In the end of the year 2007, this revolutionary technology is achieved by Japanese Yamanaka group and Thomson group of the U.S. in succession in human cell.The research of iPSCs is a fundamental research with initiative therapeutic cloning, avoid ESCs to be for a long time difficult to obtain and the problem very easily causing dispute of ethic, for stem cell medical use opens up a new way, be considered to the great discovery of stem cell field and even whole field of biology.The many scientists comprising Univ Edinburgh UK IanWilmut professor all think that inductive pluripotent stem cells is only the research direction in stem cell future.IPSCs cell not only has important theory significance for stem-cell research, and has using value in fields such as cell therapy, tissue and organ regeneration, drug screening and evaluations.
The iPS technology that present iPS technology is more initially set up is ripe a lot, but it also has a certain distance apart from myeloid-lymphoid stem cell at present, and the clinical application research of iPS cell is only limitted to theory or tentative experiment aspect.The potential of multipotential stem cell is huge, if can get rid of the potential safety hazard of iPS cell application, improves the yield of iPS cell, improves transformation efficiency further and then can bring glad tidings to more multiple large Disease.Improve the yield of iPS cell, need the improvement of many-sided condition, the culture system of selecting properly and optimization iPS cell is also vital.The cultivation of current iPS cell is mainly divided into following two kinds of training methods: cultivate without feeder layer and have feeder layer to cultivate.Now sold multifarious without feeder layer cells substratum on the market, however iPS cell in the early stage cultivation stage generally all need to depend on and can secrete them and survive in vitro and breed the feeder layer cells of necessary somatomedin.Somatomedin secreted by feeder layer cells is the conventional growing multiplication promotor of iPS cell cultures and differentiation inhibitors.
At present, the method preparing iPS cell feeder layers cell isolates mice embryonic, mice embryonic is digested to cell and inoculates.Be specially: adopt disconnected cervical approach to put to death the pregnant mouse of C57, alcohol mouse being put into 75% soaks 3-5 minute, takes out mouse, puts into aseptic super clean bench, skin and inner membrance is cut off successively with tweezers and scissors, expose internal organ, find embryo, wash 3 times in 35mm culture dish, embryo is shredded with scissors, with the tryptic digestion 15-20 minute of 0.25%, stop digestion, the centrifugal 5-10 minute of 300-600g with the substratum containing foetal calf serum; By resuspended with the DMEM in high glucose substratum of the foetal calf serum containing 8% ~ 15% for the cell precipitation obtained, Trypan Blue is adopted to carry out cell counting, then being inoculated into cell uses the gelatin solution bag of 0.1 ~ 0.5% by the Tissue Culture Flask of 0.5 ~ 2 hour, at 37 DEG C, 5%CO in advance 2cultivate in incubator.But the yield of the feeder layer cells adopting the method to obtain is lower.Therefore, the preparation method setting up a kind of efficient feeder layer cells is the key of iPS cell cultures.
Summary of the invention
In view of this, the invention provides the preparation method of a kind of digestive enzyme compositions and inoblast and feeder layer cells.This digestive enzyme compositions is by trypsinase and Dispase with after certain proportion conbined usage, and when greatly reducing digestive ferment consumption, can play better digestion effect on the contrary, fibroblastic yield is higher.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of digestive enzyme compositions, comprise trypsinase and Dispase.
Trypsin EC3.4.4.4) be the one of proteolytic enzyme, be a kind of serine protein hydrolase extracted from the pancreas of ox, sheep, pig.In vertebrates, work as digestive ferment.Be synthesized at the precursor trypsinogen of pancreas as enzyme.As pancreatic juice composition and secrete, by enteropeptidase, or activation trypsinase is decomposed in tryptic restriction, is endopeptidase, it can in Methionin in polypeptide chain and arginine residues carboxyl side cut off.It not only plays digestive ferment, and can also limit the precursor decomposing other enzymes such as chymotrypsinogen, proearboxypeptidase, phosphatide proenzyme, plays activation.Be the proteolytic enzyme that specificity is the strongest, in the amino acid range determining protein, it becomes indispensable instrument.
Dispase is a class neutral metalloprotease lytic enzyme, and compared with pancreatin, collagenase, its effect is gentleer, so can not damaging cells.In addition, Dispase also may be used for separate tissue.
The present invention studies discovery, with be used alone compared with trypsinase or Dispase, by trypsinase and Dispase with after certain proportion conbined usage, when greatly reducing digestive ferment consumption, can play better digestion effect on the contrary, fibroblastic yield is higher.
As preferably, in enzyme activity unit U, the enzyme activity ratio of trypsinase and Dispase is (1000 ~ 2000): (1 ~ 10).
In embodiments more provided by the invention, in enzyme activity unit U, the enzyme activity ratio of trypsinase and Dispase is 1250:2.2.
In other embodiments provided by the invention, in enzyme activity unit U, the enzyme activity ratio of trypsinase and Dispase is 100:1.
In other embodiments provided by the invention, in enzyme activity unit U, the enzyme activity ratio of trypsinase and Dispase is 2000:1.
Present invention also offers this digestive enzyme compositions and be prepared into the application in fibrocyte; This digestive enzyme compositions comprises trypsinase and Dispase; As preferably, in enzyme activity unit U, the enzyme activity ratio of trypsinase and Dispase is (1000 ~ 2000): (1 ~ 10); In embodiments more provided by the invention, in enzyme activity unit U, the enzyme activity ratio of trypsinase and Dispase is 1250:2.2; In other embodiments provided by the invention, in enzyme activity unit U, the enzyme activity ratio of trypsinase and Dispase is 100:1; In other embodiments provided by the invention, in enzyme activity unit U, the enzyme activity ratio of trypsinase and Dispase is 2000:1.
In embodiments more provided by the invention, inoblast is animal embryo inoblast.
In embodiments more provided by the invention, inoblast to be gestational age the be mouse embryo fibroblasts of 10 ~ 15 days.
Present invention also offers a kind of fibroblastic preparation method, comprising:
Animal embryo is shredded, adds trypsinase and Dispase digests, remove indigested tissue after stopping digestion, obtain inoblast.
As preferably, the time of digestion is 15 ~ 20min.
As preferably, embryo is embryo's trunk.In order to ensure fibroblastic purity, in the present invention, embryo used is the embryo's trunk removing head, four limbs and internal organ.
As preferably, animal embryo to be gestational age the be mice embryonic of 10 ~ 15 days.
As preferably, in enzyme activity unit U, tryptic consumption is 1000 ~ 2000U/ embryo, and the consumption of Dispase is 1 ~ 10U/ embryo.
As preferably, animal embryo is being shredded and is adding between trypsinase and Dispase digest, also comprising and add trypsinase and continue to cut 1 ~ 3min.
Present invention also offers a kind of preparation method of feeder layer cells, comprising:
Animal embryo is shredded, adds trypsinase and Dispase digests, remove indigested tissue after stopping digestion, obtain inoblast;
Adopt ametycin to be processed into fibrocyte, through digestion, cell cultures, obtain feeder layer cells.
As preferably, inoblast is that P3 is for inoblast.
As preferably, cell cultures be 37 DEG C, volume fraction is the CO of 5% 224h is cultivated under condition.
In embodiments more provided by the invention, the condition of ametycin process is: 37 DEG C, volume fraction is the CO of 5% 2ametycin process 2 ~ 3h is adopted under condition.
In embodiments more provided by the invention, after adopting ametycin to be processed into fibrocyte, adopt tryptic digestion 1 ~ 2min.
As preferably, the cell concn of cell cultures is (1 ~ 10) × 10 7/ L.
Preferably, the cell concn of cell cultures is 5.0 × 10 7/ L.
The invention provides the preparation method of a kind of digestive enzyme compositions and inoblast and feeder layer cells.This digestive enzyme compositions comprises trypsinase and Dispase.The present invention at least has one of following advantage:
1, the present invention is by trypsinase and Dispase with after certain proportion conbined usage, and when greatly reducing digestive ferment consumption, can play better digestion effect, fibroblastic yield is higher;
2, only retain the trunk of embryo in inoblast sepn process, the fibroblastic purity obtained is higher.
Accompanying drawing explanation
Fig. 1 shows P 0for the form of MEF;
Fig. 2 shows the preparation flow of feeder layer cells;
Fig. 3 shows the Dynamic Curve figure of feeder layer cells.
Embodiment
The invention discloses the preparation method of a kind of digestive enzyme compositions and inoblast and feeder layer cells, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
Terminological interpretation:
Feeder layer cells: refer to some specific cells (as the cell that granulosa cell, inoblast, Epithelium Cells etc. are supported in vitro), the cell monolayer of gained after mitotic block agent (conventional mitomycin) process.Available MEF (mouse embryo fibroblasts) the treated acquisition of feeder layer cells major part.
Inoblast (fibroblast): the main cellular being loose connective tissue, is differentiated by the mesenchymal cell (mesenchymalcell) of embryonic stage.Inoblast is comparatively large, and profile is clear, mostly is the fusiform of projection or the flats structure of star, and its nucleus is in the oval of rule, and kernel greatly and obviously.According to difference in functionality active state, cell can be divided into inoblast and fibrocyte, Fibroblast Function activity is vigorous, tenuigenin is addicted to weakly alkaline, the obvious protein synthesis of tool and secretory activity, under certain condition, it can realize with fibrocellular mutual conversion.The reparation of inoblast to cytopathy in various degree, necrosis and tissue defect and bone wound has very important effect.
In the preparation method of digestive enzyme compositions provided by the invention and inoblast and feeder layer cells, laboratory animal used, digestive ferment, reagent etc. all can be buied by market.In embodiments of the present invention, tryptic enzyme activity is 250U/mg, and the enzyme activity of Dispase is 1.1U/mg.Mouse type: C57BL/6.
Below in conjunction with embodiment, set forth the present invention further:
The preparation of embodiment 1 embryo fibroblast
The pregnant mouse of C57 of 13 days is bought from Guangdong Medical Lab Animal Center, adopt disconnected cervical approach to put to death mouse, alcohol mouse being put into 75% soaks 3-5 minute, takes out mouse, put into aseptic super clean bench, cut off skin with a set of clean tweezers and scissors, the tweezers that transducer set is clean and scissors, cut off skin inner membrance, expose internal organ, find embryo, a general 8-10 embryo/pregnant mouse, wash 3 times in 35mm culture dish.
The aseptic apparatus that transducer set is clean, two curved tweezers, tear fetal membrane, remove the head of embryo, four limbs and internal organ, remaining organizing are shredded as far as possible, about 1mm 3size, continues to cut 1 minute with the trypsinase of 1mL0.125%, then adds the trypsinase of 2mL0.25% and the Dispase of 2mL0.1% digests 15 minutes at culture dish; During this period, took out culture dish every 5 minutes, with rifle head piping and druming mixing 3-5 time; Stop digestion with the substratum containing foetal calf serum, filter with 200 object screen clothes, remove indigested tissue, the centrifugal 5-10 minute of 300-600g.
By resuspended with the DMEM in high glucose substratum of the foetal calf serum containing 10% for the cell precipitation obtained, adopt trypan blue staining to carry out cell counting, the quantity of the MEF cell of acquisition is 6.7 × 10 6/ pregnant mouse.
The preparation of embodiment 2 embryo fibroblast
The pregnant mouse of C57 of 10 days is bought from Guangdong Medical Lab Animal Center, adopt disconnected cervical approach to put to death mouse, alcohol mouse being put into 75% soaks 3-5 minute, takes out mouse, put into aseptic super clean bench, cut off skin with a set of clean tweezers and scissors, the tweezers that transducer set is clean and scissors, cut off skin inner membrance, expose internal organ, find embryo, a general 8-10 embryo/pregnant mouse, wash 3 times in 35mm culture dish.
The aseptic apparatus that transducer set is clean, two curved tweezers, tear fetal membrane, remove the head of embryo, four limbs and internal organ, remaining organizing are shredded as far as possible, about 1mm 3size, continues to cut 2 minutes with the trypsinase of 1mL0.125%, then adds the trypsinase of 3.2mL0.25% and the Dispase of 0.9mL0.1% digests 18 minutes at culture dish; During this period, took out culture dish every 5 minutes, with rifle head piping and druming mixing 3-5 time; Stop digestion with the substratum containing foetal calf serum, filter with 200 object screen clothes, remove indigested tissue, the centrifugal 5-10 minute of 300-600g.
By resuspended with the DMEM in high glucose substratum of the foetal calf serum containing 10% for the cell precipitation obtained, adopt trypan blue staining to carry out cell counting, the quantity of the MEF cell of acquisition is 6.29 × 10 6/ pregnant mouse.
The preparation of embodiment 3 embryo fibroblast
The pregnant mouse of C57 of 15 days is bought from Guangdong Medical Lab Animal Center, adopt disconnected cervical approach to put to death mouse, alcohol mouse being put into 75% soaks 3-5 minute, takes out mouse, put into aseptic super clean bench, cut off skin with a set of clean tweezers and scissors, the tweezers that transducer set is clean and scissors, cut off skin inner membrance, expose internal organ, find embryo, a general 8-10 embryo/pregnant mouse, wash 3 times in 35mm culture dish.
The aseptic apparatus that transducer set is clean, two curved tweezers, tear fetal membrane, remove the head of embryo, four limbs and internal organ, remaining organizing are shredded as far as possible, about 1mm 3size, continues to cut 3 minutes with the trypsinase of 1mL0.125%, then adds the trypsinase of 1.6mL0.25% and the Dispase of 9mL0.1% digests 20 minutes at culture dish; During this period, took out culture dish every 5 minutes, with rifle head piping and druming mixing 3-5 time; Stop digestion with the substratum containing foetal calf serum, filter with 200 object screen clothes, remove indigested tissue, the centrifugal 5-10 minute of 300-600g.
By resuspended with the DMEM in high glucose substratum of the foetal calf serum containing 10% for the cell precipitation obtained, adopt trypan blue staining to carry out cell counting, the quantity of the MEF cell of acquisition is 5.4 × 10 6/ pregnant mouse.
The preparation of embodiment 4 feeder layer cells
MEF cell embodiment 1 obtained is inoculated into uses the gelatin solution bag of 0.1 ~ 0.5% by the Tissue Culture Flask of 0.5 ~ 2 hour, at 37 DEG C, 5%CO in advance 2cultivate in incubator.Every day observation of cell form: the embryo fibroblast (MEF cell) after adherent in irregular trilateral, fan-shaped or fusiformis (as Fig. 1).After 5 ~ 7 days, MEF cell is gone down to posterity.
By P3 for MEF cell when growing to about 90%, substratum is changed into the nutrient solution of ametycin containing 5% ~ 20%, is placed in 37 DEG C, volume fraction is the CO of 5% 22 ~ 3 hours are acted in incubator; Wash 3-5 time by PBS solution and remove residual ametycin; Add the tryptic digestion 1-2 minute of 0.25%; Digestion is stopped, centrifugal 5 minutes of 1000rpm with the perfect medium of MEF cell; Abandon supernatant, add MEF cell culture medium re-suspended cell precipitation, adjustment cell concn is 5.0 × 10 7/ L, every hole adds 1mL cell suspension inoculation in 24 orifice plates, 37 DEG C, volume fraction is the CO of 5% 2cultivate in incubator.Cultivate after 24 hours, obtain feeder layer cells, can be used for inoculation iPS cell.Prepare flow graph and see Fig. 2.
Adopt CCK-8 method to detect the vigor of feeder layer cells, detection method is: by the MEF cell after ametycin process with after the tryptic digestion of 0.25%, according to 2 × 10 3/ hole is inoculated in 96 orifice plates, and 4 multiple holes, inoculate 28 holes, every hole 100 μ l cell suspension altogether; After cultivating 24h, get 4 multiple holes and add 10 μ lCCK-8 solution continuation cultivation 1-4 hour, at 450nm wavelength, its absorbance is detected at place, and reference wavelength is 600-650nm; Get the detection that absorbance is carried out in 4 multiple holes later every day, continuous detecting 7 days, is depicted as growth curve by the absorbance of gained.The vigor detecting the feeder layer cells of cultivating 1-7 days by CCK8 method is shown in Fig. 3.
Feeder layer cells vigor in inoculation 3 days is higher as seen from Figure 3, and after 3 days, cell viability significantly declines, and compared with first 3 days, difference has significant.Ametycin Inhibit proliferaton phase DNA copies, the propagation of T suppression cell, is feeder layer cells, can be supplied to iPS cytotrophy by the cell of the known acquisition of above-mentioned test-results, for inducing iPS cell proliferation.
The separation and Culture of comparative example 1 l cell
Disconnected cervical approach is adopted to put to death the pregnant mouse of C57, alcohol mouse being put into 75% soaks 3-5 minute, take out mouse, put into aseptic super clean bench, skin and inner membrance is cut off successively with tweezers and scissors, expose internal organ, find embryo, wash 3 times in 35mm culture dish, embryo is shredded with scissors, with the tryptic digestion 15-20 minute of 0.25%, digestion is stopped with the substratum containing foetal calf serum, the centrifugal 5-10 minute of 300-600g, by resuspended with the DMEM in high glucose substratum of the foetal calf serum containing 8% ~ 15% for the cell precipitation obtained, Trypan Blue is adopted to carry out cell counting, the quantity of the MEF cell obtained is 2.9 × 10 6/ pregnant mouse.
Then MEF cell is inoculated into and uses the gelatin solution bag of 0.1 ~ 0.5% by the Tissue Culture Flask of 0.5 ~ 2 hour in advance, at 37 DEG C, 5%CO 2cultivate in incubator.
The preparation of comparative example 2 embryo fibroblast
The pregnant mouse of C57 of 13 days is bought from Guangdong Medical Lab Animal Center, adopt disconnected cervical approach to put to death mouse, alcohol mouse being put into 75% soaks 3-5 minute, takes out mouse, put into aseptic super clean bench, cut off skin with a set of clean tweezers and scissors, the tweezers that transducer set is clean and scissors, cut off skin inner membrance, expose internal organ, find embryo, a general 8-10 embryo/pregnant mouse, wash 3 times in 35mm culture dish.
The aseptic apparatus that transducer set is clean, two curved tweezers, tear fetal membrane, remove the head of embryo, four limbs and internal organ, remaining organizing are shredded as far as possible, about 1mm 3size, the trypsinase with 0.125% continues to cut 1-3 minute, then the trypsinase adding 4mL0.25% digests 15 minutes at culture dish; During this period, took out culture dish every 5 minutes, with rifle head piping and druming mixing 3-5 time; Stop digestion with the substratum containing foetal calf serum, filter with 200 object screen clothes, remove indigested tissue, the centrifugal 5-10 minute of 300-600g.
By resuspended with the DMEM in high glucose substratum of the foetal calf serum containing 10% for the cell precipitation obtained, adopt trypan blue staining to carry out cell counting, the quantity of the MEF cell of acquisition is 4.74 × 10 6/ pregnant mouse.
The preparation of comparative example 3 embryo fibroblast
The pregnant mouse of C57 of 13 days is bought from Guangdong Medical Lab Animal Center, adopt disconnected cervical approach to put to death mouse, alcohol mouse being put into 75% soaks 3-5 minute, takes out mouse, put into aseptic super clean bench, cut off skin with a set of clean tweezers and scissors, the tweezers that transducer set is clean and scissors, cut off skin inner membrance, expose internal organ, find embryo, a general 8-10 embryo/pregnant mouse, wash 3 times in 35mm culture dish.
The aseptic apparatus that transducer set is clean, two curved tweezers, tear fetal membrane, remove the head of embryo, four limbs and internal organ, remaining organizing are shredded as far as possible, about 1mm 3size, the trypsinase with 0.125% continues to cut 1-3 minute, then the Dispase adding 4mL0.1% digests 15 minutes at culture dish; During this period, took out culture dish every 5 minutes, with rifle head piping and druming mixing 3-5 time; Stop digestion with the substratum containing foetal calf serum, filter with 200 object screen clothes, remove indigested tissue, the centrifugal 5-10 minute of 300-600g.
By resuspended with the DMEM in high glucose substratum of the foetal calf serum containing 10% for the cell precipitation obtained, adopt trypan blue staining to carry out cell counting, the quantity of the MEF cell of acquisition is 1.56 × 10 6/ pregnant mouse.
Conclusion:
From above-mentioned test-results, the quantity of the MEF cell that embodiment of the present invention method obtains is 6.7 × 10 6/ pregnant mouse, and the quantity of the MEF cell adopting existing technology of preparing to obtain is 2.9 × 10 6/ pregnant mouse, its quantity is well below method provided by the invention, and difference has statistical significance.Meanwhile, when consumption greatly reduces, the digestion effect of digestive enzyme compositions provided by the invention is significantly better than the digestion effect being used alone trypsinase or Dispase, visible, and trypsinase and Dispase conbined usage can be produced digestion effect better.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (10)

1. a digestive enzyme compositions, is characterized in that, comprises trypsinase and Dispase.
2. digestive enzyme compositions according to claim 1, is characterized in that, in enzyme activity unit U, the enzyme activity ratio of described trypsinase and described Dispase is (1000 ~ 2000): (1 ~ 10).
3. digestive enzyme compositions as claimed in claim 1 or 2 is being prepared into the application in fibrocyte.
4. a fibroblastic preparation method, is characterized in that, comprising:
Animal embryo is shredded, adds trypsinase and Dispase digests, remove indigested tissue after stopping digestion, obtain inoblast.
5. preparation method according to claim 4, is characterized in that, the time of described digestion is 15 ~ 20min.
6. preparation method according to claim 4, is characterized in that, described embryo is embryo's trunk.
7. preparation method according to claim 4, is characterized in that, described animal embryo to be gestational age the be mice embryonic of 10 ~ 15 days.
8. preparation method according to claim 7, is characterized in that, in enzyme activity unit U, described tryptic consumption is 1000 ~ 2000U/ embryo, and the consumption of described Dispase is 1 ~ 10U/ embryo.
9. a preparation method for feeder layer cells, is characterized in that, comprising:
Animal embryo is shredded, adds trypsinase and Dispase digests, remove indigested tissue after stopping digestion, obtain inoblast;
Adopt inoblast described in ametycin process, through digestion, cell cultures, obtain feeder layer cells.
10. preparation method according to claim 9, is characterized in that, cell cultures be 37 DEG C, volume fraction is the CO of 5% 224h is cultivated under condition.
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