CN109938009A - A kind of umbilical cord tissue transport liquid and preparation method - Google Patents
A kind of umbilical cord tissue transport liquid and preparation method Download PDFInfo
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- CN109938009A CN109938009A CN201910216217.9A CN201910216217A CN109938009A CN 109938009 A CN109938009 A CN 109938009A CN 201910216217 A CN201910216217 A CN 201910216217A CN 109938009 A CN109938009 A CN 109938009A
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Abstract
The present invention relates to a kind of umbilical cord tissue transport liquid and preparation method thereof, and transport liquid includes DMEM culture solution: 100ml；Penicillin: 0.014~0.026g；Aminoglycoside antibiotics: 0.014~0.026g；Polyene antibiotic: 0.49~0.51mg.Preparation method includes step 1, prepares transport liquid by part；Weigh the drug in component when making every part of transport liquid according to quantity respectively；Drug is dissolved in DMEM culture solution by step 2, obtains transport liquid；Step 3, by the packing of every part of transport liquid, vacuum seal and cryo-conservation.It joined polyene antibiotic, aminoglycoside antibiotics and penicillin in transport liquid of the present invention, can targetedly prevent umbilical cord tissue that by microorganism infection, there is better antibacterial effect during transportation.
Description
Technical field
The present invention relates to cells to transport liquid, and in particular to a kind of umbilical cord tissue transport liquid and preparation method.
Background technique
Umbilical cord is to link structure between fetus and placenta.Umbilical cord tissue medically has very wide application prospect.Example
As stored abandoned umbilical cord tissue cell abundant and group can be provided for the gene therapy for realizing individuation and cell therapy
Knit resources reserve.There is also there is umbilical cord mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) in umbilical cord tissue, it is
Finger is present in one of neonatal umbilical cord tissue versatile stem cell, it can be divided into many kinds of histocytes, has wide
Potential applicability in clinical practice.
The biggest obstacle of storage and transport umbilical cord tissue is, during umbilical cord tissue transports in vitro, in order to subsequent
Culture and application, tissue/cell are required in the overall process of preparation culture in germ-free condition.Guarantee is collected
Umbilical cord is able to maintain sterile in transport and storage process, and conventionally employed umbilical cord tissue is immersed in PBS collection liquid saves.
But the preparation of PBS is complicated, when voluntarily preparing, will increase a large amount of workload on being prepared on a large scale, if purchase finished product, because
It is expensive, higher operating costs.
Summary of the invention
In view of the deficiencies of the prior art, the invention discloses a kind of umbilical cord tissue transport liquid and preparation methods.
The technical solution adopted in the present invention is as follows:
A kind of umbilical cord tissue transport liquid, every part of transport liquid include:
DMEM culture solution: 100ml；
Penicillin: 0.014~0.026g；
Aminoglycoside antibiotics: 0.014~0.026g；
Polyene antibiotic: 0.49~0.51mg.
Its further technical solution are as follows:
DMEM culture solution；100ml；
Penicillin: 0.018~0.022g；
Aminoglycoside antibiotics: 0.018~0.022g；
Polyene antibiotic: 0.5~0.51mg.
Its further technical solution are as follows: the polyene antibiotic is amphotericin B or nystatin.Preferably,
The polyene antibiotic is amphotericin B.
Its further technical solution are as follows: the aminoglycoside antibiotics is streptomysin, gentamicin, kanamycins
It is one or several kinds of.Preferred: the aminoglycoside antibiotics is streptomysin.
Its further technical solution are as follows: the penicillin is penicillin G sodium salt.
Polyene antibiotic: polyene antibiotic is also known as polyene macrolide antibiotics (polyene macrolide
Antibiotics, PEM are to contain conjugated double bond and antifungal antibiotic as produced by certain actinomyces and fungi.All present
The feature of polyenic compounds, polyene antibiotic mainly inhibit the cholesterol of fungal cell membrane, such as fungi and mycoplasma is thin
After birth.
Aminoglycoside antibiotics: aminoglycoside antibiotics (Aminoglycosides) is by amino sugar and amino ring
The tobramycin antibiotic that alcohol is formed by connecting by oxygen bridge mainly inhibits the effect of bacterium the synthesis of bacterio protein, harm
Hinder the synthesis of initial composite object, Induction of bacterial resultant fault albumen and the release for suppressing synthetic proteins, so as to cause bacterium
It is dead.
Penicillin: penicillin (Penicillin) is one kind of antibiotic, refers to and contains penam in molecule, can destroy carefully
The cell wall of bacterium and from the breeding period of bacterial cell bactericidal effect a kind of antibiotic, be the antibiosis by being extracted in Penicillium notatum
Element.
The preparation method of liquid is transported according to umbilical cord tissue described in any one as above, comprising:
Step 1 transports liquid by part preparation；Weigh the drug in component when making every part of transport liquid according to quantity respectively；
Drug is dissolved in DMEM culture solution by step 2, obtains transport liquid；
Step 3 dispenses every part of transport liquid 125ml liquid storage bottle, and every bottle of liquid storage bottle storage 100ml transports liquid.It twists later
Tight bottle cap, is sealed with sealed membrane, after being packed into valve bag, is reloaded into vacuum seal pocket, and vacuum seal is carried out.
Its further technical solution is, in the step 2, is first by the mass percentage concentration that drug is dissolved in 40ml
In 0.9% DMEM culture solution；Filtered fluid is obtained by filtration using 0.22 μm of filter later；Then filtered fluid is diluted to 500ml
Obtain transport liquid；It is finally dispensed, every bottle of 100ml, vacuum seal saves.
Step made above is both needed to operate in gnotobasis.
Beneficial effects of the present invention are as follows:
(1) polyene antibiotic, aminoglycoside antibiotics and penicillin be joined in transport liquid of the present invention,
It can targetedly prevent umbilical cord tissue that by microorganism infection, there is better antibacterial effect during transportation.
(2) present invention is using based on DMED culture solution, if various antibiotic, pass through and control containing for each ingredient
Amount, can during transportation play a very good protection to umbilical cord tissue, maintain the morphosis and biology of umbilical cord tissue
Activity extends the holding time of umbilical cord tissue, improves the survival rate of umbilical cord tissue.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the invention is clearly and completely described.
Umbilical cord tissue used in following embodiment is derived from healthy puerpera's normal labor or postcesarean umbilical cord.
Examples 1 to 3 is to have made a transport liquid, can also be made according to ratio less than a (such as moiety, four points
Portion etc.) or more parts of transport liquid.The transport liquid component of Examples 1 to 3 is shown in Table 1
Table 1
| Ingredient | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| DMEM culture solution (ml) | 100 | 100 | 100 |
| Penicillin G sodium salt (g) | 0.01 | 0.014 | 0.026 |
| Streptomysin (g) | 0.02 | 0.026 | 0.014 |
| Amphotericin B (mg) | 0.5 | 0.49 | 0.51 |
The preparation method of umbilical cord tissue transport liquid in above-described embodiment 1~3, specifically includes the following steps:
Step 1 weighs each drug according to the component weight of each drug；
Drug is dissolved in 40ml, in the DMEM culture solution that concentration is 0.9% by step 2, obtains just solution processed；
Filtered fluid is obtained by filtration using 0.22 μm of filter is carried out in first solution processed by step 3；Filtering solution is diluted to again
500ml obtains transport liquid.
Step 4 dispenses transport liquid, dispenses specifically, the liquid liquid storage bottle of 125ml will be transported, each liquid storage bottle is equipped with
The transport liquid of 100ml.
Step 5, vacuum and low temperature save, and the transport liquid after packing is tightened bottle cap, is sealed with sealed membrane, are packed into self-styled
It after bag, is reloaded into vacuum seal pocket, carries out vacuum seal.The transport liquid of sealing is finally placed in -20 DEG C of preservations.
It is by the method that umbilical cord tissue is stored in above-mentioned umbilical cord tissue transport liquid:
Step A, the transport lyolysis saved at a temperature of being placed on -20 DEG C is frozen to 4 DEG C.
Step B, by healthy puerpera's normal labor or postcesarean umbilical cord, it is clear that 4 DEG C of umbilical cord is placed in after in vitro at once
It is tentatively saved in washing lotion.
Step C, umbilical cord tissue will be taken under the umbilical cord aseptic condition tentatively saved, is rinsed with umbilical cord cleaning solution.Later by navel
It is placed in 4 DEG C obtained in step A of transport liquid with tissue.
Step D, the transport liquid cooling equipped with umbilical cord tissue is stored in simulate in the cold chain transportation case of 4 DEG C of environment and was transported
Journey.
Step E, step D treated umbilical cord tissue is taken out, culture, observes the culture situation of umbilical cord tissue cell.
According to the above store method, it is as follows that a reference examples are set:
Reference examples 1:
Prepare the sample of reference examples 1:
Step A, four groups are taken, every group of 100 parts of transport liquid, the transport lyolysis saved at a temperature of being placed on -20 DEG C later is frozen
To 4 DEG C.
Step B, by healthy puerpera's normal labor or postcesarean umbilical cord, it is clear that 4 DEG C of umbilical cord is placed in after in vitro at once
It is tentatively saved in washing lotion.
Step C, umbilical cord tissue will be taken under the umbilical cord aseptic condition tentatively saved, removes endodermis, is rushed with umbilical cord cleaning solution
It washes, is divided into four groups；Every group includes 100 parts.Wherein three groups of solution are respectively placed in 4 DEG C obtained in step A of transport liquid later
In, another set is placed in DMED culture solution.
Step D, to transportational process is simulated in 4 DEG C of cold chain transportation casees of transport liquid equipped with umbilical cord tissue, 5 hours are saved.
Step E, by step D, treated that umbilical cord tissue is finally takes out to umbilical cord tissue, culture, and observation umbilical cord tissue is thin
The culture situation of born of the same parents, living cell counting ratio.
Reference examples 1 the results are shown in Table 2:
Table 2
| DMEM culture solution | Embodiment 1 transports liquid | Embodiment 2 transports liquid | Embodiment 3 transports liquid | |
| Transport sum | 100 | 100 | 100 | 100 |
| Survive sum | 48 | 97 | 94 | 95 |
| Survival rate | 48% | 97% | 94% | 95% |
As shown in table 2, after umbilical cord tissue saves in transport liquid, result is deposited than what is saved in DMEM culture solution
Motility rate is high.Wherein the condition in embodiment 1 is optimal configuration method.
Embodiment 4~6 is to have made a transport liquid, can also be made according to ratio less than a (such as moiety, four points
Portion etc.) or more parts of transport liquid.The transport liquid component of embodiment 4~6 is shown in Table 3
Table 3
| Ingredient | Embodiment 4 | Embodiment 5 | Embodiment 6 |
| DMEM culture solution (ml) | 100 | 100 | 100 |
| Penicillin G sodium salt (g) | 0.024 | 0.014 | 0.026 |
| Gentamicin (g) | 0.022 | 0.014 | 0.026 |
| Nystatin (mg) | 0.502 | 0.49 | 0.51 |
Umbilical cord tissue in above-described embodiment 4~6 transports the preparation method of liquid and umbilical cord tissue is stored in umbilical cord group
The method knitted in transport liquid is identical as Examples 1 to 3.
According to the above store method, it is as follows that a reference examples are set:
Reference examples 2:
Prepare the sample of reference examples 2:
Step A, four groups are taken, every group of 100 parts of transport liquid, the transport lyolysis saved at a temperature of being placed on -20 DEG C later is frozen
To 4 DEG C.
Step B, by healthy puerpera's normal labor or postcesarean umbilical cord, it is clear that 4 DEG C of umbilical cord is placed in after in vitro at once
It is tentatively saved in washing lotion.
Step C, umbilical cord tissue will be taken under the umbilical cord aseptic condition tentatively saved, removes endodermis, is rushed with umbilical cord cleaning solution
It washes, is divided into four groups；Every group includes 100 parts.Four groups of solution are respectively placed in 4 DEG C obtained in step A of transport liquid later.
Step D, transportational process is simulated to 4 DEG C of cold chain transportation casees of transport liquid equipped with umbilical cord tissue, saves 6 hours.
Step E, step D treated umbilical cord tissue is taken out, culture, observes the culture situation of umbilical cord tissue cell, meter
Number live cell fraction.
Reference examples 2 the results are shown in Table 4:
Table 4
| DMEM culture solution | Embodiment 4 transports liquid | Embodiment 5 transports liquid | Embodiment 6 transports liquid | |
| Transport sum | 100 | 100 | 100 | 100 |
| Survive sum | 45 | 94 | 92 | 93 |
| Survival rate | 45% | 94% | 92% | 93% |
As shown in table 4, after umbilical cord tissue saves in transport liquid, result is deposited than what is saved in DMEM culture solution
Motility rate is high.Wherein the condition in embodiment 1 is optimal configuration method.
Above description is explanation of the invention, is not intended to limit the invention, and limited range of the present invention is referring to right
It is required that the present invention can make any type of modification without prejudice to basic structure of the invention.
Claims (9)
1. a kind of umbilical cord tissue transports liquid, which is characterized in that every part of transport liquid includes:
DMEM culture solution: 100ml；
Penicillin: 0.014~0.026g；
Aminoglycoside antibiotics: 0.014~0.026g；
Polyene antibiotic: 0.49~0.51mg.
2. umbilical cord tissue according to claim 1 transports liquid, it is characterised in that: every part of transport liquid includes:
DMEM culture solution；100ml；
Penicillin: 0.018~0.022g；
Aminoglycoside antibiotics: 0.018~0.022g；
Polyene antibiotic: 0.5~0.51mg.
3. umbilical cord tissue according to claim 1 or 2 transports liquid, it is characterised in that: the polyene antibiotic is two
Property mycin B or nystatin.
4. umbilical cord tissue according to claim 3 transports liquid, it is characterised in that: the polyene antibiotic is anphotericin
B。
5. umbilical cord tissue according to claim 1 or 2 transports liquid, it is characterised in that: the aminoglycoside antibiotics
For the one or several kinds of streptomysin, gentamicin, kanamycins.
6. umbilical cord tissue according to claim 5 transports liquid, it is characterised in that: the aminoglycoside antibiotics is strepto-
Element.
7. umbilical cord tissue according to claim 1 or 2 transports liquid, it is characterised in that: the penicillin is novocillin
Salt.
8. the preparation method of umbilical cord tissue transport liquid according to claim 1 or 2, characterized by comprising:
Step 1 transports liquid by part preparation；Weigh the drug in component when making every part of transport liquid according to quantity respectively；
Drug is dissolved in DMEM culture solution by step 2, obtains transport liquid；
Step 3 dispenses every part of transport liquid 125ml liquid storage bottle, and every bottle of liquid storage bottle storage 100ml transports liquid.Bottle is tightened later
Lid, is sealed with sealed membrane, after being packed into valve bag, is reloaded into vacuum seal pocket, and vacuum seal is carried out.
9. preparation method according to claim 8, which is characterized in that in the step 2, first drug will be dissolved in 40ml
Mass percentage concentration be 0.9% DMEM culture solution in；Filtered fluid is obtained by filtration using 0.22 μm of filter later；Then it incited somebody to action
Filtrate is diluted to 500ml and obtains transport liquid；It is finally dispensed, every bottle of 100ml, vacuum seal saves.
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| CN110810399A (en) * | 2019-11-21 | 2020-02-21 | 康妍葆(北京)干细胞科技有限公司 | Cell transportation protective solution, preparation method and application |
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Application publication date: 20190628 |