CN109913433A - People's umbilical cord Wharton jelly tissue digestion enzyme and preparation method thereof - Google Patents
People's umbilical cord Wharton jelly tissue digestion enzyme and preparation method thereof Download PDFInfo
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- CN109913433A CN109913433A CN201910214305.5A CN201910214305A CN109913433A CN 109913433 A CN109913433 A CN 109913433A CN 201910214305 A CN201910214305 A CN 201910214305A CN 109913433 A CN109913433 A CN 109913433A
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- umbilical cord
- wharton jelly
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- cord wharton
- clostridiopetidase
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Abstract
The invention discloses a kind of people's umbilical cord Wharton jelly tissue digestion enzymes and preparation method thereof, it is calculated in weight percent, contain following raw material: hyaluronidase 16.0~23.5%, DNA enzymatic 0.16~0.23%, clostridiopetidase A 3.2~4.7%, DMEM46.9~63.9%, gentamicin 0.8~1.2%, penicillin G sodium salt 16.0~23.5%.After hyaluronidase, DNA enzymatic, clostridiopetidase A are dissolved in DMEM, filtered with 0.22 μm of filter, it is rear that gentamicin and penicillin G sodium salt is added, it is frozen in -20 DEG C of refrigerators.By the above-mentioned means, the present invention can efficiently digest umbilical cord Wharton jelly tissue, umbilical cord Wharton jelly mescenchymal stem cell is quickly obtained, and can effectively prevent cell contamination, biological effect is significant, has broad application prospects.
Description
Technical field
The present invention relates to technical field of cell separation, more particularly to a kind of people's umbilical cord Wharton jelly tissue digestion enzyme and its system
Preparation Method.
Background technique
Mescenchymal stem cell is derived from mesoblastic stem cell, is a kind of the dry of the multi-lineage potential of non-haematological origin
Cell.Since its immunogenicity is weak, and can to Various Tissues Proliferation, Differentiation, therefore, have become tissue engineering technique field and
Ideal seed cell source in cellular transplantation therapy.Huatong plastic mescenchymal stem cell derives from neonatal umbilical cord, belongs to medical treatment
Discarded tissue does not generate in privation and damage to donor, and from a wealth of sources, therefore be easier compared to mesenchymal stem cell
It is widely applied in clinic.Huatong plastic tissue is the gel-shaped interstitial between umbilical cord arteriovenous, contains more collagenous fibres, glue
Protoenzyme can hydrolyze the collagen fiber structure of huatong plastic tissue, but not logical glue mesenchymal stem cells cause to damage to China.
Currently, separation obtains the method for umbilical cord Wharton jelly mescenchymal stem cell mainly organized adherent method and enzyme from umbilical cord
Two class of digestion method, enzyme digestion include single enzyme process (clostridiopetidase A), two enzymes method (clostridiopetidase A, trypsase) and three enzyme process (collagens
Enzyme, hyaluronidase, trypsase) digestion method.
It organizes adherent method easy to operate, but suffers from the drawback that being unfavorable for huatong plastic mescenchymal stem cell dissociates, and causes navel
Waste with tissue；The cell of acquisition is not pure, Chang Hanyou various kinds of cell；Huatong plastic, smudge cells DNA increase culture medium
Viscosity；Cell is adherent slowly, influences cultivation cycle；It is easy to cause germ contamination.
Enzyme digestion there is no unified standard in the selection and digestion time of the type and quantity of enzyme, be primarily present following ask
Topic: the thorough digestion time of tissue block is long；Digestive ferment type is single, cannot thoroughly digest hyaluronic acid, collagenous fibres；It is simple to increase
The dosage of enzyme can destroy free cell surface protein such as adhesion factor out, cell viability is made to be affected with adherent ability.
Summary of the invention
The invention mainly solves the technical problem of providing a kind of people's umbilical cord Wharton jelly tissue digestion enzymes, can reduce digestion
Digestion is not thorough in the process, reduces damage to mescenchymal stem cell, and control cell is lost, and avoids that cell is not pure and bacterium is dirty
Dye problem.
In order to solve the above technical problems, one technical scheme adopted by the invention is that: a kind of people's umbilical cord Wharton jelly group is provided
Digestive ferment is knitted, it is calculated in weight percent, contain following raw material: hyaluronidase 16.0~23.5%, DNA enzymatic 0.16~
0.23%, clostridiopetidase A 3.2~4.7%, DMEM46.9~63.9%, gentamicin 0.8~1.2%, penicillin G sodium salt 16.0~
23.5%.
A kind of the step of human umbilical tissue digestive ferment, preparation method, is as follows:
S1, count by weight percentage, takes raw material as follows: hyaluronidase 16.0~23.5%, DNA enzymatic 0.16~
0.23%, clostridiopetidase A 3.2~4.7%, DMEM46.9~63.9%, gentamicin 0.8~1.2%, penicillin G sodium salt 16.0~
23.5%.
S2, hyaluronidase, DNA enzymatic and clostridiopetidase A are first dissolved in DMEM, is then obtained by filtration using 0.22 μm of filter
Mixed liquor.
S3, S2 mixed liquor is taken, adds gentamicin and penicillin G sodium salt, frozen in -20 DEG C of refrigerators.
The beneficial effects of the present invention are: the present invention is low in cost, preparation is simple, and effect efficiently, saves convenient transportation.
Specific embodiment
Combined with specific embodiments below the present invention is made further to explain.
Embodiment 1
A kind of human umbilical tissue digestive ferment takes raw material hyaluronidase 100mg, DNA enzymatic 1mg, clostridiopetidase A 20mg,
DMEM200ml, gentamicin 5ml, penicillin G sodium salt 100mg.By weight percentage: hyaluronidase 23.5%, DNA enzymatic
0.23%, clostridiopetidase A 4.7%, DMEM46.9%, gentamicin 1.2%, penicillin G sodium salt 23.5%, method and step is as follows:
S1: weighing hyaluronidase 100mg, DNA enzymatic 1mg, clostridiopetidase A 20mg in aseptic safe super-clean bench, and DMEM is added
In culture medium 200ml；
S2: mixed liquor made from S1 is passed through to 0.22 μm of strainer filtering, and adds gentamicin 5ml, penicillin G sodium salt
100mg, be placed in -20 DEG C it is spare.
Embodiment 2
A kind of human umbilical tissue digestive ferment takes raw material hyaluronidase 100mg, DNA enzymatic 1mg, clostridiopetidase A 20mg,
DMEM400ml, gentamicin 5ml, penicillin G sodium salt 100mg.By weight percentage: hyaluronidase 16.0%, DNA enzymatic
0.16%, clostridiopetidase A 3.2%, DMEM63.9%, gentamicin 0.8%, penicillin G sodium salt 16.0%
Preparation method step such as embodiment 1.
Embodiment 3
A kind of human umbilical tissue digestive ferment takes raw material hyaluronidase 100mg, DNA enzymatic 1mg, clostridiopetidase A 20mg,
DMEM300ml, gentamicin 5ml, penicillin G sodium salt 100mg.By weight percentage: hyaluronidase 19.0%, DNA enzymatic
0.19%, clostridiopetidase A 3.8%, DMEM57.0%, gentamicin 0.9%, penicillin G sodium salt 19.0%
The preparation method is the same as that of Example 1.
Embodiment 4
Monthly output healthy newborn umbilical cord about 30cm is taken fully, sterile storage is in D-Hanks liquid, 2-6 DEG C of preservation；
Umbilical cord remained blood is washed away with balanced salt solution in super-clean bench, umbilical cord is cut into 4cm size segment with operating scissors,
Each segment is taken, umbilical cord outer membrane is cut off in operating scissors stringer, carefully free 2 umbilical arteries and 1 umbilical vein and is abandoned, is taken blood
Huatong plastic tissue between pipe and between blood vessel and outer membrane cuts into the big fine grained chippings of about 1mm3, after balanced salt solution rinses, leaching
In Examples 1 to 3 product, 37 DEG C of constant temperature digestions are set 18 hours；
Observation digestion situation, has no tissue block, and for digestive juice at thick, 2500rpm is centrifuged 10min, discards upper layer supernatant,
Remaining viscous liquid uses 37 DEG C of 0.25% trypsase constant temperature digestion 30 minutes again；
2500rpm is centrifuged 10min after digestion, abandons supernatant, adds DMEM/F12 culture medium and fetal calf serum, keep fetal calf serum dense
Degree reaches 20%, in 37 DEG C, 5%CO2 CMC model；
Cell is carried out on the 4th day in the 2nd day beginning adherent growth with the DMEM/F12 complete medium containing 20% fetal calf serum
Change liquid；
It changes the liquid once within every 2 to 3 days later, when cell fusion reaches 80-90%, carries out secondary culture；
Supernatant is abandoned, PBS washes cell, and 0.25% trypsin digestion 5min, 1000rpm centrifugation carries out cell passage；
It is cultivated after passage with the DMEM/F12 culture medium containing 10% fetal calf serum, carries out changing liquid within every 3 days.
Using the tissue digestion enzyme can make huatong plastic tissue pieces digest it is more thorough, tissue enzyme mixation stickiness compared with
It is low, and general digestive ferment can make tissue enzyme mixation stickiness higher, be not easy to filter, the mixed liquor for filtering same volume can
Excessive filter can be needed, waste is be easy to cause.And cell is also easy to be not easy to filter out together with tissue mucus
Come.After the filtering of tissue digestion enzyme cell at single dispersion in the medium, be easy it is adherent, and cell it is adherent after divided quickly
Proliferation is split, common digestive juice has a large amount of mucus, prevents cell adherent and the division of cell.
Claims (3)
1. a kind of people's umbilical cord Wharton jelly tissue digestion enzyme, which is characterized in that it is calculated in weight percent, contain following raw material: thoroughly
Bright matter acid enzyme 16.0~23.5%, DNA enzymatic 0.16~0.23%, clostridiopetidase A 3.2~4.7%, DMEM46.9~63.9%, celebrating are big
Mycin 0.8~1.2%, penicillin G sodium salt 16.0~23.5%.
2. the preparation method of people's umbilical cord Wharton jelly tissue digestion enzyme according to claim 1, which is characterized in that by hyalomitome
It is dissolved in DMEM after sour enzyme, DNA enzymatic, clostridiopetidase A mixing, and is filtered with 0.22 μm of filter, adds gentamicin, novocillin
Salt freezes in -20 DEG C of refrigerators.
3. the preparation method of people's umbilical cord Wharton jelly tissue digestion enzyme according to claim 1, which is characterized in that all operations
Process operates in an aseptic environment.
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| CN201910214305.5A CN109913433A (en) | 2019-03-20 | 2019-03-20 | People's umbilical cord Wharton jelly tissue digestion enzyme and preparation method thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106867981A (en) * | 2017-04-14 | 2017-06-20 | 青岛青春派生物科技有限公司 | A kind of human umbilical tissue digestive ferment and preparation method thereof |
| CN107043746A (en) * | 2017-04-14 | 2017-08-15 | 青岛青春派生物科技有限公司 | A kind of human umbilical tissue digestion method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106867981A (en) * | 2017-04-14 | 2017-06-20 | 青岛青春派生物科技有限公司 | A kind of human umbilical tissue digestive ferment and preparation method thereof |
| CN107043746A (en) * | 2017-04-14 | 2017-08-15 | 青岛青春派生物科技有限公司 | A kind of human umbilical tissue digestion method |
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Application publication date: 20190621 |