CN104894088A - Digestive enzyme composition and application thereof, and isolated culture method of umbilical epithelial cells - Google Patents
Digestive enzyme composition and application thereof, and isolated culture method of umbilical epithelial cells Download PDFInfo
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
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Abstract
The invention relates to the technical field of cell isolated culture, particularly a digestive enzyme composition and application thereof, and an isolated culture method of umbilical epithelial cells. The digestive enzyme composition comprises hyaluronidase, collagenase VIII and pancreatin. The umbilical epithelial cell primary isolation adopts the hyaluronidase, collagenase VIII and pancreatin for combined digestion. The digestive enzyme composition has the advantages of mild properties, low cell trauma and high enzymolysis rate and greatly enhances the epithelial cell isolation efficiency.
Description
Technical field
The present invention relates to cellular segregation culture technique field, particularly digestive enzyme compositions and application thereof, the epithelial isolation cultivation method of umbilical cord.
Background technology
Umbilical cord is the tubular structure connecting fetus and placenta, is originally that the handle elongated portion yolk sac and allantois by amnion winding is formed.In umbilical cord, arteriovenous is the vascularization by allantois, and namely the blood vessel of yolk sac forms omphalomescenteric artery and omphalomescenteric vein.When yolk sac and vascular deterioration thereof, Umbilical artery and umbilical vein get up with regard to prosperity, can see the mesenchyme of loose glue in these gaps.Umbilical cord be parent and fetus blood between carry out CO
2and O
2, the place that exchanges of metabolic waste and nutritive substance.The refuse that fetus produces is transported to placenta by Umbilical artery, and umbilical vein is by O
2transport to fetus with nutritive substance from placenta, finally by uterine veins, the metabolic waste from fetus is transported.
And certain hormone and antibody etc. also transfer fetus by umbilical cord to from parent, but operating mechanism is not yet clear.Be separated by vitro culture and cultivate umbilical cord epithelial cell, can to the epithelial multiplication characteristic of umbilical cord, some hormone between mother and baby and the running of antibody and mechanism etc. are studied.Meanwhile, isolate people's umbilical cord epithelial cell, also can be applicable to the recovery application of human body burned skin, there is far-reaching medical research meaning.
At present, and the related art scheme that unmanned umbilical cord epithelial cell is separated, with reference to the epithelial cell separation scheme of its hetero-organization, all adopt tissue mass cell culture to be separated epithelial cell.
Number of patent application be the invention of CN201210113304.X report a kind of Goat Mammary Epithelial Cells build system, method, the isolation and purification of Goat Mammary Epithelial Cells is:
(1) separation of Goat Mammary Epithelial Cells
(1) breast tissue be separated with Gestation period goat is selected, the DPBS solution put into after sterilization and clean containing penicillin and Streptomycin sulphate washs, then fat and reticular tissue is removed, leave white granular acinar tissue block, put into the DPBS solution washing 2 times containing penicillin and Streptomycin sulphate again, finally proceed to not containing the DPBS solution washing one time of penicillin and Streptomycin sulphate;
(2) appropriate tissue block is put into centrifuge tube, drip number and bleed moistening clearly, be cut into 1mm
3fritter, the shred rear homogeneous microstructure appropriate with pipettor sucking-off coats culture dish, and spacing keeps 1cm
2left and right;
(3) 37 DEG C, 5%CO
2, back-off dries up cultivation 6 ~ 12h under saturated humidity, period is not stirred;
(4) above-mentioned (3) back-off dries up after cultivation terminates, and adds 2mL mammary epithelial cell nutrient solution, in 37 DEG C, 5%CO
2, just put cultivation under saturated humidity;
(5) after inoculation tissue block 24h, add mammary epithelial cell nutrient solution to 4 ~ 6mL, after inoculation tissue block 72h, add mammary epithelial cell nutrient solution to 8 ~ 10mL; Observe once every 3d later, notice that observation of cell climbs out of situation and pollution condition, and take the circumstances into consideration to change liquid; The composition of described mammary epithelial cell nutrient solution is: DMEM/F12+10%FBS+1%ITS+10ng/mLEGF;
(2) purifying of Goat Mammary Epithelial Cells
(6) when cell mixing adherent in culture dish reach 80 ~ 90% converge time, draw substratum, DPBS washs 2 times;
(7) mammary epithelial cell Digestive system is added in 37 DEG C, 5%CO
2, digest under saturated humidity; The composition of described mammary epithelial cell Digestive system comprises 0.02%EDTA and 0.25%Trypsin;
(8) period constantly observes, and becomes circle and when being about to come off, rocking rapidly the culture dish cell being about to come off under existing Digestive system band, and exhaust with pipettor when most of inoblast;
(9) above-mentioned Digestive system is again added in 37 DEG C, 5%CO rapidly
2, digest under saturated humidity;
(10) period constantly observes, and when most of epithelioid cell is about to come off, adds mammary epithelial cell and stops nutrient solution termination digestion; The ratio of going down to posterity remains 1: 2; Described mammary epithelial cell stop buffer composition is: DMEM/F12+10%FBS;
(11) (7) ~ (11) step is repeated, until inoblast is removed clean.
Number of patent application is that the invention of CN201410176073.6 provides the separation of a kind of prostata tissue and sets up the method for primary epithelial cells and/or mesenchymal cell, and concrete steps comprise:
The first step: mouse prostate leaf texture shredded, with the washing of primary cell culture liquid, transfers to the tissue shredded in centrifuge tube, and centrifugal;
Second step: transfer in T-25 culturing bottle with the resuspended tissue of primary cell culture liquid, cultivate 1 ~ 2 day in 37 DEG C of incubators, after at the bottom of tissue block sticks to bottle, pour out nutrient solution and rejoin primary cell culture liquid and cultivate, after 2 ~ 3 days, mouse prostate epithelium and mesenchymal cell go out from tissue block surrounding growth;
3rd step: fully digest the cell grown with pancreatin, centrifugal collecting cell also transfers them to that another is new in the culturing bottle of primary culture medium, obtain mouse prostate and organize primary epithelial cells and mesenchymal cell: or, the type of various biological cells and tissues block is distinguished under inverted microscope, with cell scraper, mesenchymal cell and tissue block are wiped off or epithelial cell and tissue block are wiped off, after primary culture medium washing, the cell grown fully is digested with pancreatin, centrifugal collecting cell also transfers them to that another is new in the culturing bottle of primary culture medium, obtain mouse prostate and organize primary epithelial cells or mesenchymal cell.
But the acquisition methods original cuiture time of above-mentioned existing tissue epithelial cell is long, obtain total cellular score low, the low-doped inoblast of cell purity.Therefore, set up a kind of quick, that cell yield is high, cell purity is high umbilical cord epithelial cell separation method to have important practical significance.
Summary of the invention
In view of this, the invention provides digestive enzyme compositions and application thereof, the epithelial isolation cultivation method of umbilical cord.The method adopts Unidasa, collagenase VIII and pancreatin simultaneous digestion, and digestive enzyme compositions character is gentle, and little to cell injury, enzymolysis speed is high, and epithelial cell separation efficiency promotes greatly.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of digestive enzyme compositions, comprise Unidasa, collagenase VIII and pancreatin.
In the present invention, the primary separation of umbilical cord epithelial cell adopts Unidasa, collagenase VIII and pancreatin simultaneous digestion, and this digestive enzyme compositions character is gentle, and little to cell injury, enzymolysis speed is high, and epithelial cell separation efficiency promotes greatly.
As preferably, Unidasa, collagenase VIII are (5 ~ 30) with the mass ratio of pancreatin: (1 ~ 10): (1 ~ 10).
In embodiments more provided by the invention, Unidasa, collagenase VIII are 5:1:1 with the mass ratio of pancreatin.
In other embodiments provided by the invention, Unidasa, collagenase VIII are 5:5:10 with the mass ratio of pancreatin.
In other embodiments provided by the invention, Unidasa, collagenase VIII are 10:10:1 with the mass ratio of pancreatin.
In other embodiments provided by the invention, Unidasa, collagenase VIII are 30:1:5 with the mass ratio of pancreatin.
In embodiments more provided by the invention, the specification of Unidasa is 300U/mg.
In embodiments more provided by the invention, the specification of collagenase VIII is 125CDU/mg.
In embodiments more provided by the invention, the specification of pancreatin is 1800U/mg.
Present invention also offers this digestive enzyme compositions and be separated the application in umbilical cord epithelial cell; This digestive enzyme compositions comprises Unidasa, collagenase VIII and pancreatin; As preferably, Unidasa, collagenase VIII are (5 ~ 30) with the mass ratio of pancreatin: (1 ~ 10): (1 ~ 10); In embodiments more provided by the invention, Unidasa, collagenase VIII are 5:1:1 with the mass ratio of pancreatin; In other embodiments provided by the invention, Unidasa, collagenase VIII are 5:5:10 with the mass ratio of pancreatin; In other embodiments provided by the invention, Unidasa, collagenase VIII are 10:10:1 with the mass ratio of pancreatin; In other embodiments provided by the invention, Unidasa, collagenase VIII are 30:1:5 with the mass ratio of pancreatin.
Present invention also offers the epithelial isolation cultivation method of a kind of umbilical cord, comprise the steps:
Adopt digestive enzyme compositions to digest umbilical cord surface overcoats film, acquisition umbilical cord epithelial cell is carried out cell cultures; This digestive enzyme compositions comprises Unidasa, collagenase VIII and pancreatin.
As preferably, Unidasa, collagenase VIII are (5 ~ 30) with the mass ratio of pancreatin: (1 ~ 10): (1 ~ 10).
In embodiments more provided by the invention, Unidasa, collagenase VIII are 5:1:1 with the mass ratio of pancreatin.
In other embodiments provided by the invention, Unidasa, collagenase VIII are 5:5:10 with the mass ratio of pancreatin.
In other embodiments provided by the invention, Unidasa, collagenase VIII are 10:10:1 with the mass ratio of pancreatin.
In other embodiments provided by the invention, Unidasa, collagenase VIII are 30:1:5 with the mass ratio of pancreatin.
In embodiments more provided by the invention, the specification of Unidasa is 300U/mg, and being mixed with concentration is 0.05% ~ 0.30%.
In embodiments more provided by the invention, the specification of collagenase VIII is 125CDU/mg, and being mixed with concentration is 0.01% ~ 0.10%.
In embodiments more provided by the invention, the specification of pancreatin is 1800U/mg, and being mixed with concentration is 0.01% ~ 0.10%.
In embodiments more provided by the invention, according to the area of umbilical cord surface overcoats film, with mL/cm
2meter, the add-on of digestive ferment is: the add-on of Unidasa is 1mL/cm
2, the add-on of collagenase VIII is 1mL/cm
2, the add-on of pancreatin is 1mL/cm
2.
As preferably, the substratum of cell cultures is the DMEM/F12 substratum containing 10%FBS adding EGF.EGF can promote cell proliferation, shortens culture cycle.
As preferably, the mass percentage of EGF is 0.01 ~ 1%.
In embodiments more provided by the invention, the mass percentage of EGF is 0.1%.
As preferably, cell cultures is at 36 ~ 38 DEG C, 5%CO
2degrees of fusion 80% is cultured under condition.
In embodiments more provided by the invention, cell cultures is at 37 DEG C, 5%CO
2degrees of fusion 80% is cultured under condition.
In embodiments more provided by the invention, the epithelial density of umbilical cord is (1 ~ 5) × 10
5/ mL.
As preferably, the epithelial density of umbilical cord is 1 × 10
5/ mL.
In embodiments more provided by the invention, the epithelial inoculum size of umbilical cord is 1mL/cm
2.
As preferably, the substratum of cell cultures adopts gelatin bag quilt.The epithelial original cuiture of umbilical cord, adopt gelatin bag by after Tissue Culture Dish, increase the adherent rate of cell, reduce the time of original cuiture, increase the total cellular score that original cuiture obtains.
As preferably, the temperature of digestion is 36 ~ 38 DEG C, and the time of digestion is 0.5 ~ 3h.
In embodiments more provided by the invention, the temperature of digestion is 37 DEG C, and the time of digestion is 1h.
In embodiments more provided by the invention, cell cultures is: the DMEM/F12 substratum containing 10%FBS getting umbilical cord epithelial cell interpolation EGF is resuspended, and adjustment cell density is (1 ~ 5) × 10
5/ mL, by 1mL/cm
2inoculum size be seeded to cell ware, in 37 DEG C, 5%CO
22h is cultivated under condition; Draw culture supernatant and be transferred to the Tissue Culture Dish of gelatin bag quilt in 37 DEG C, 5%CO
2cultivate under condition, go down to posterity when ware bottom growth degrees of fusion is 80% until cell.
As preferably, the epithelial density of umbilical cord is 1 × 10
5/ mL.
The invention provides digestive enzyme compositions and application thereof, the epithelial isolation cultivation method of umbilical cord.This digestive enzyme compositions comprises Unidasa, collagenase VIII and pancreatin.The present invention at least has one of following advantage:
In the present invention, the primary separation of umbilical cord epithelial cell adopts Unidasa, collagenase VIII and pancreatin simultaneous digestion, and this digestive enzyme compositions character is gentle, and little to cell injury, enzymolysis speed is high, and epithelial cell separation efficiency promotes greatly;
The present invention adds EGF in cell culture medium, can promote cell proliferation, shortens culture cycle;
The present invention adopt gelatin bag by after Tissue Culture Dish, the adherent rate of cell can be increased, reduce time of original cuiture, increase the total cellular score that original cuiture obtains.
Accompanying drawing explanation
Fig. 1 shows the epithelial growth curve of the different algebraically of two kinds of methods.
Embodiment
The invention discloses digestive enzyme compositions and application thereof, the epithelial isolation cultivation method of umbilical cord, 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.
In digestive enzyme compositions provided by the invention and application thereof, the epithelial isolation cultivation method of umbilical cord, agents useful for same, instrument all can be buied by market.Wherein, the specification of Unidasa is 5g/ bottle, 300U/mg; The specification of Collagenase V III is 1g/ bottle, 125CDU/mg; The specification of pancreatin is 10g/ bottle, 1800U/mg; The specification of neutral protease is 2g/ bottle, 100000U/ bottle; The specification of collagenase IV is 5g/ bottle, 625000CDU/ bottle.
Below in conjunction with embodiment, set forth the present invention further:
The epithelial primary separation and ientification of embodiment 1 people umbilical cord
(1) the gelatin bag quilt of culture dish
Take gelatin powder (Sigma), be made into 0.1% (W/V) solution with deionized water, 37 DEG C of water-baths are dissolved, and the filter screen of filtered while hot 0.22 μm, be positioned over 4 DEG C for subsequent use.
Get the 0.1% gelatin solution rewarming prepared, according to the size of Tissue Culture Dish area, add 0.3mL/cm
20.1% gelatin solution, is evenly paved with at the bottom of ware, in 37 DEG C of cell culture incubators, place 24h, then the whole gelatin sucking-offs in ware is discarded, and cell ware gelatin bag is done.
(2) the epithelial primary separation of people's umbilical cord
Umbilical cord after collection hospital pregnant woman throws aside postpartum.Aseptically get a umbilical cord, umbilical cord surface bloodstain is cleaned with the phosphate buffered saline buffer (PBS) that 100mL is aseptic, then use 100mL 75% ethanol disinfection umbilical cord surface, finally use the aseptic phosphate buffered saline buffer of 100mL (PBS) alcohol and bloodstain to be cleaned up.
Strip the outermost tunic on umbilical cord surface, outer membrane is shredded, according to outer membrane area, add 1mL/cm
2the associating enzyme liquid of 0.25% Unidasa+0.05% collagenase VIII+0.05% pancreatin is to after membrane digestion (37 DEG C) 1h, by 100 μm of filter screen filtration, the centrifugal 5min of filtrate 300g, remove supernatant liquor, add DMEM/F12+10%FBS+0.1%EGF cell culture medium resuspended, regulate cell density to be 1 × 10
5/ mL, by 1mL/cm
2density cells liquid is seeded to cell ware and is positioned over 37 DEG C, 5%CO
2cultivate 2 hours in cell culture incubator.
After 2 hours, absorption culture supernatant is transferred to Tissue Culture Dish (the gelatin bag that first part obtains is by culture dish) and is positioned over 37 DEG C, 5%CO
2cultivate in cell culture incubator.Until cell when ware bottom growth degrees of fusion is 80%, can go down to posterity.
(3) people's umbilical cord is epithelial goes down to posterity
Remove cell culture medium supernatant, after washing 3 times with phosphate buffered saline buffer (PBS), add 0.02mL/cm
20.1% pancreatin+0.02%EDTA Digestive system digestion 5min, enzymolysis is stopped with 8 times of DMEM/F12+10%FBS+0.1%EGF cell culture mediums to Digestive system, 300g centrifugal 5min, DMEM/F12+10%FBS+0.1%EGF cell culture medium is resuspended, and cell density is 1 × 10
5/ mL, according to culture dish area, 1mL/cm
2be inoculated in the culture dish not having gelatin bag quilt.
The epithelial primary separation and ientification of embodiment 2 people umbilical cord
(1) the gelatin bag quilt of culture dish
Take gelatin powder (Sigma), be made into 0.1% (W/V) solution with deionized water, 37 DEG C of water-baths are dissolved, and the filter screen of filtered while hot 0.22 μm, be positioned over 4 DEG C for subsequent use.
Get the 0.1% gelatin solution rewarming prepared, according to the size of Tissue Culture Dish area, add 0.3mL/cm
20.1% gelatin solution, is evenly paved with at the bottom of ware, in 37 DEG C of cell culture incubators, place 24h, then the whole gelatin sucking-offs in ware is discarded, and cell ware gelatin bag is done.
(2) the epithelial primary separation of people's umbilical cord
Umbilical cord after collection hospital pregnant woman throws aside postpartum.Aseptically get a umbilical cord, umbilical cord surface bloodstain is cleaned with the phosphate buffered saline buffer (PBS) that 100mL is aseptic, then use 100mL 75% ethanol disinfection umbilical cord surface, finally use the aseptic phosphate buffered saline buffer of 100mL (PBS) alcohol and bloodstain to be cleaned up.
Strip the outermost tunic on umbilical cord surface, outer membrane is shredded, according to outer membrane area, add 1mL/cm
2the associating enzyme liquid of 0.05% Unidasa+0.05% collagenase VIII+0.1% pancreatin is to after membrane digestion (36 DEG C) 3h, by 100 μm of filter screen filtration, the centrifugal 5min of filtrate 300g, remove supernatant liquor, add DMEM/F12+10%FBS+0.01%EGF cell culture medium resuspended, regulate cell density to be 2 × 10
5/ mL, by 1mL/cm
2density cells liquid is seeded to cell ware and is positioned over 37 DEG C, 5%CO
2cultivate 2 hours in cell culture incubator.
After 2 hours, absorption culture supernatant is transferred to Tissue Culture Dish (the gelatin bag that first part obtains is by culture dish) and is positioned over 37 DEG C, 5%CO
2cultivate in cell culture incubator.Until cell when ware bottom growth degrees of fusion is 80%, can go down to posterity.
(3) people's umbilical cord is epithelial goes down to posterity
Remove cell culture medium supernatant, after washing 3 times with phosphate buffered saline buffer (PBS), add 0.02mL/cm
20.1% pancreatin+0.02%EDTA Digestive system digestion 5min, enzymolysis is stopped with 8 times of DMEM/F12+10%FBS+0.1%EGF cell culture mediums to Digestive system, 300g centrifugal 5min, DMEM/F12+10%FBS+0.1%EGF cell culture medium is resuspended, and cell density is 1-2 × 10
5/ mL, according to culture dish area, 1mL/cm
2be inoculated in the culture dish not having gelatin bag quilt.
The epithelial primary separation and ientification of embodiment 3 people umbilical cord
(1) the gelatin bag quilt of culture dish
Take gelatin powder (Sigma), be made into 0.1% (W/V) solution with deionized water, 37 DEG C of water-baths are dissolved, and the filter screen of filtered while hot 0.22 μm, be positioned over 4 DEG C for subsequent use.
Get the 0.1% gelatin solution rewarming prepared, according to the size of Tissue Culture Dish area, add 0.3mL/cm
20.1% gelatin solution, is evenly paved with at the bottom of ware, in 37 DEG C of cell culture incubators, place 24h, then the whole gelatin sucking-offs in ware is discarded, and cell ware gelatin bag is done.
(2) the epithelial primary separation of people's umbilical cord
Umbilical cord after collection hospital pregnant woman throws aside postpartum.Aseptically get a umbilical cord, umbilical cord surface bloodstain is cleaned with the phosphate buffered saline buffer (PBS) that 100mL is aseptic, then use 100mL 75% ethanol disinfection umbilical cord surface, finally use the aseptic phosphate buffered saline buffer of 100mL (PBS) alcohol and bloodstain to be cleaned up.
Strip the outermost tunic on umbilical cord surface, outer membrane is shredded, according to outer membrane area, add 1mL/cm
20.10% Unidasa+0.10% collagenase VIII+0.01% pancreatin is to after membrane digestion (38 DEG C) 0.5h, by 100 μm of filter screen filtration, the centrifugal 5min of filtrate 300g, remove supernatant liquor, add DMEM/F12+10%FBS+1%EGF cell culture medium resuspended, regulate cell density to be 5 × 10
5/ mL, by 1mL/cm
2density cells liquid is seeded to cell ware and is positioned over 37 DEG C, 5%CO
2cultivate 2 hours in cell culture incubator.
After 2 hours, absorption culture supernatant is transferred to Tissue Culture Dish (the gelatin bag that first part obtains is by culture dish) and is positioned over 37 DEG C, 5%CO
2cultivate in cell culture incubator.Until cell when ware bottom growth degrees of fusion is 80%, can go down to posterity.
(3) people's umbilical cord is epithelial goes down to posterity
Remove cell culture medium supernatant, after washing 3 times with phosphate buffered saline buffer (PBS), add 0.02mL/cm
20.1% pancreatin+0.02%EDTA Digestive system digestion 5min, enzymolysis is stopped with 8 times of DMEM/F12+10%FBS+0.1%EGF cell culture mediums to Digestive system, 300g centrifugal 5min, DMEM/F12+10%FBS+0.1%EGF cell culture medium is resuspended, and cell density is 1-2 × 10
5/ mL, according to culture dish area, 1mL/cm
2be inoculated in the culture dish not having gelatin bag quilt.
The epithelial primary separation and ientification of embodiment 4 people umbilical cord
(1) the gelatin bag quilt of culture dish
Take gelatin powder (Sigma), be made into 0.1% (W/V) solution with deionized water, 37 DEG C of water-baths are dissolved, and the filter screen of filtered while hot 0.22 μm, be positioned over 4 DEG C for subsequent use.
Get the 0.1% gelatin solution rewarming prepared, according to the size of Tissue Culture Dish area, add 0.3mL/cm
20.1% gelatin solution, is evenly paved with at the bottom of ware, in 37 DEG C of cell culture incubators, place 24h, then the whole gelatin sucking-offs in ware is discarded, and cell ware gelatin bag is done.
(2) the epithelial primary separation of people's umbilical cord
Umbilical cord after collection hospital pregnant woman throws aside postpartum.Aseptically get a umbilical cord, umbilical cord surface bloodstain is cleaned with the phosphate buffered saline buffer (PBS) that 100mL is aseptic, then use 100mL 75% ethanol disinfection umbilical cord surface, finally use the aseptic phosphate buffered saline buffer of 100mL (PBS) alcohol and bloodstain to be cleaned up.
Strip the outermost tunic on umbilical cord surface, outer membrane is shredded, according to outer membrane area, add 1mL/cm
2the associating enzyme liquid of 0.3% Unidasa+0.01% collagenase VIII+0.05% pancreatin is to after membrane digestion (37 DEG C) 1h, by 100 μm of filter screen filtration, the centrifugal 5min of filtrate 300g, remove supernatant liquor, add DMEM/F12+10%FBS+0.1%EGF cell culture medium resuspended, regulate cell density to be 1 × 10
5/ mL, by 1mL/cm
2density cells liquid is seeded to cell ware and is positioned over 37 DEG C, 5%CO
2cultivate 2 hours in cell culture incubator.
After 2 hours, absorption culture supernatant is transferred to Tissue Culture Dish (the gelatin bag that first part obtains is by culture dish) and is positioned over 37 DEG C, 5%CO
2cultivate in cell culture incubator.Until cell when ware bottom growth degrees of fusion is 80%, can go down to posterity.
(3) people's umbilical cord is epithelial goes down to posterity
Remove cell culture medium supernatant, after washing 3 times with phosphate buffered saline buffer (PBS), add 0.02mL/cm
20.1% pancreatin+0.02%EDTA Digestive system digestion 5min, enzymolysis is stopped with 8 times of DMEM/F12+10%FBS+0.1%EGF cell culture mediums to Digestive system, 300g centrifugal 5min, DMEM/F12+10%FBS+0.1%EGF cell culture medium is resuspended, and cell density is 1 × 10
5/ mL, according to culture dish area, 1mL/cm
2be inoculated in the culture dish not having gelatin bag quilt.
Embodiment 5 umbilical cord epithelial cell separation rate calculates
Choosing 15 umbilical cords, be divided into 5 groups at random, is experimental group and control group, and the difference of each group is to use different digestive ferment, and all the other experimental procedures are identical with embodiment 1.
Experimental group 1:0.25% Unidasa+0.05% collagenase VIII+0.05% pancreatin;
Experimental group 2:0.05% Unidasa+0.05% collagenase VIII+0.1% pancreatin;
Experimental group 3:0.10% Unidasa+0.10% collagenase VIII+0.01% pancreatin;
Experimental group 4:0.3% Unidasa+0.01% collagenase VIII+0.05% pancreatin;
Control group 1:0.10% neutral protease+0.05% collagenase IV.
Experiment concrete operations are as follows:
Umbilical cord after collection hospital pregnant woman throws aside postpartum.Aseptically get a umbilical cord, umbilical cord surface bloodstain is cleaned with the phosphate buffered saline buffer (PBS) that 100mL is aseptic, then use 100mL 75% ethanol disinfection umbilical cord surface, finally use the aseptic phosphate buffered saline buffer of 100mL (PBS) alcohol and bloodstain to be cleaned up.
Strip the outermost tunic on umbilical cord surface, outer membrane is shredded, according to outer membrane area, add digestive ferment (experimental group 1:0.25% Unidasa+0.05% collagenase VIII+0.05% pancreatin; Experimental group 2:0.05% Unidasa+0.05% collagenase VIII+0.1% pancreatin; Experimental group 3:0.10% Unidasa+0.10% collagenase VIII+0.01% pancreatin; Experimental group 4:0.3% Unidasa+0.01% collagenase VIII+0.05% pancreatin; Control group 1:0.10% neutral protease+0.05% collagenase IV) to after membrane digestion 1h, by 100 μm of filter screen filtration, the centrifugal 5min of filtrate 300g, removes supernatant liquor, add DMEM/F12+10%FBS+0.1%EGF cell culture medium resuspended, regulate cell density to be 1 × 10
5/ mL, by 1mL/cm
2density cells liquid is seeded to cell ware and is positioned over 37 DEG C, 5%CO
2cultivate 2 hours in cell culture incubator.
After 2 hours, draw culture supernatant, cell counting A is carried out to supernatant liquor, is transferred to Tissue Culture Dish (gelatin bag quilt) and is positioned over 37 DEG C, 5%CO
224h is cultivated in cell culture incubator, remove supernatant liquor, after trypsin digestion cell, add 8 times of DMEM/F12+10%FBS+0.1%EGF cell culture mediums to Digestive system and stop digestion, cell counting B is carried out to nutrient solution, calculate epithelial cell adherent rate, calculation formula is: epithelial cell adherent rate=B/A × 100%.
Two groups of epithelial cell separation rate measurement results see the following form:
Table 1 original cuiture epithelial cell adherent rate
Experimental group 1 ~ 4 is higher than control group 1 adherent rate as can be seen from Table 1, and when experimental group 1 ~ 4 is inoculated, isolated total cellular score is also higher than control group 1, and the adherent rate of parallel laboratory test is also more consistent.Prove that experimental group 1 ~ 4 enzyme solution separation efficiency used is high, little to cell injury.
The epithelial cell proliferation curve comparison of the different cultural method of embodiment 6
CCK-8 method is adopted to detect the epithelial proliferation activity of umbilical cord cultivated.Choose 6 umbilical cords, be divided into two groups at random, experimental group 1 is separated according to the method for example 1 and cultivates epithelial cell, reaches P10 generation always.Experimental group 2 does not adopt in the culture dish of gelatin bag quilt and substratum does not add EGF, and all the other experimental techniques, according to example 1, reach P10 generation always.Two groups of cells being collected in P1, P5, P10 respectively, inoculating cell in 96 orifice plates, 2000/hole.At 37 DEG C, 5%CO
2cultured continuously 7d in incubator.At 1d, 3d, 5d, 7d, cell is detected respectively.10 μ L staining agents are added, 37 DEG C, 5%CO in gaging hole to be checked
2cultivate 2h.Survey the light absorption value at 450nm place by microplate reader, each sample does 6 repetitions, sees Fig. 1.
As seen from Figure 1, each generation cell in experimental group 1 all has higher growth curve compared to experimental group 2, and experimental group 2 generation is higher, and propagation is more tending towards slow, proves that EGF in experimental group 1 and gelatin bag are greatly promoted and maintain the vigor of cell proliferation.
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 Unidasa, collagenase VIII and pancreatin.
2. digestive enzyme compositions according to claim 1, is characterized in that, described Unidasa, described collagenase VIII are (5 ~ 30) with the mass ratio of described pancreatin: (1 ~ 10): (1 ~ 10).
3. digestive enzyme compositions according to claim 1, is characterized in that, described Unidasa, described collagenase VIII are 5:1:1 with the mass ratio of described pancreatin.
4. according to any one of claims 1 to 3, digestive enzyme compositions is being separated the application in umbilical cord epithelial cell.
5. the epithelial isolation cultivation method of umbilical cord, is characterized in that, comprise the steps:
Adopt digestive enzyme compositions according to any one of claims 1 to 3 to digest umbilical cord surface overcoats film, acquisition umbilical cord epithelial cell is carried out cell cultures.
6. isolation cultivation method according to claim 5, is characterized in that, the substratum of described cell cultures is the DMEM/F12 substratum containing 10%FBS adding EGF.
7. isolation cultivation method according to claim 6, is characterized in that, the mass percentage of described EGF is 0.01 ~ 1%.
8. isolation cultivation method according to claim 5, is characterized in that, described cell cultures is at 36 ~ 38 DEG C, 5%CO
2degrees of fusion 80% is cultured under condition.
9. isolation cultivation method according to claim 5, is characterized in that, the substratum of described cell cultures adopts gelatin bag quilt.
10. the isolation cultivation method according to any one of claim 5 to 9, is characterized in that, the temperature of described digestion is 36 ~ 38 DEG C, and the time of digestion is 0.5 ~ 3h.
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