CN107114356A - A kind of placenta and umbilical cord cells protect liquid - Google Patents
A kind of placenta and umbilical cord cells protect liquid Download PDFInfo
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- CN107114356A CN107114356A CN201610625635.XA CN201610625635A CN107114356A CN 107114356 A CN107114356 A CN 107114356A CN 201610625635 A CN201610625635 A CN 201610625635A CN 107114356 A CN107114356 A CN 107114356A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The invention provides a kind of placenta and umbilical cord cells protection liquid; the protection liquid mainly forms mycoplasma inhibitor and aminoglycoside antibiotics with MEM-α culture medium aqueous dissolutions; MEM-α culture mediums the aqueous solution described in per mL dissolves the 30mg of mycoplasma inhibitor 10, the 240U of the aminoglycoside antibiotics 180 respectively, and the concentration of the MEM-α culture medium aqueous solution is 10 15mg/mL.The protection liquid that the present invention is provided can not only effectively kill or suppress the formation of mycoplasma; prevent placenta cells or umbilical cord cells from infecting; and can be that placenta or umbilical cord cells provide a stable osmotic pressure environment by adding protective ingredient; effectively simulation human body environment, improves cells survival rate, while nutritional ingredient can be provided for storage and transport in the cell short time; improve the immune performance of cell; cell infection is prevented, the viability of cell is effectively improved, extends cell haulage time.
Description
Technical field
The invention belongs to placenta and umbilical cord cells Techniques of preserving field, more particularly to a kind of placenta and umbilical cord cells protection
Liquid.
Background technology
In recent years, preserve placenta stem-cell and the upsurge of cord blood stem cell goes up year by year, most people is it is known that profit
The many benefits of disease in the blood system etc. can be cured with cord blood stem cell, but the biomedicine for placenta stem-cell is led
The great utility in domain is but known little about it.Preserve placenta and mainly extracted from the placenta of preservation and contained in mescenchymal stem cell, placenta
Some mescenchymal stem cells enrich very much, and this mescenchymal stem cell belongs to multipotential stem cell, with powerful multiplication capacity and
Multi-lineage potential, can cultivate the various histoorgans and nervous system of development adult body, in addition, placenta mesenchyma stem cell
Diseases range is treated than wide, for example, treats Cardial or cerebral vascular diseases, the nervous system disease, liver diseases, bone tissue disease, angle
A variety of myopathies such as film loss, empyrosis, myopathy；Similarly, many people preserve Cord blood mainly from the Cord blood of preservation
Umbilical cord mesenchymal stem cells are extracted, umbilical cord mesenchymal stem cells have multi-lineage potential, hematopoiesis support and promote stem cell to plant
Enter, immunoregulation and the features such as self-replacation, umbilical cord mesenchymal stem cells are damaged for histoorgan caused by aging and lesion
Wound repairs preferable seed cell.Therefore, because placenta stem-cell and cord blood stem cell are for the important for the treatment of human body diseases
Property, increasing people starts storage umbilical cord mesenchymal stem cells and placenta stem-cell in case the possible period of want or need in future.
Storage umbilical cord mesenchymal stem cells and placenta stem-cell generally comprise collection, transport, handing-over, detection, separation, jelly
Deposit, recover, all multi-steps such as original cuiture and Secondary Culture, need to transport to before separating after umbilical cord and placenta collection, join
With three steps of detection, need to deposit longer time in practical operation.Umbilical cord and placenta are once gathered, departing from original body
Interior environment, the activity of its cell can decline rapidly in a short time, and this directly affects its isolated placenta cells and navel
Quality and vigor with cell, therefore, the problem of how maintaining the activity of in vitro placenta and umbilical cord cells and become primary solution.
Time, temperature, osmotic pressure etc..
Placenta and umbilical cord can effectively be preserved to realize, existing patent publication No. discloses for CN105028388A
Protection liquid and preparation method thereof, dextran, sodium chloride, calcium chloride, potassium chloride, glucose and human blood are contained in the protection liquid
The solution of albumin composition, suitable Conservation environment is provided for cell, but the protection liquid prepares complicated, and stability of solution
Difference, can not substantially simulate during human body environment, and cell cryopreservation osmotic pressure and freeze temperature and can result in cell and deposit
Activity decrease during storage, active material loss is very fast, and cell is easy to apoptosis during longer-term storage, therefore, being badly in need of exploitation
One kind can extend cell storage life and can effectively ensure that cytoactive, prevent placenta and the umbilical cord cells protection of Apoptosis
Liquid.
The content of the invention
Although can preserve placenta or umbilical cord cells to solve protection liquid of the prior art, this protection liquid is matched somebody with somebody
System is complicated, and stability of solution is poor, can not substantially simulate during human body environment, and cell cryopreservation osmotic pressure and freeze
Temperature can result in activity decrease during cell storage, and active material loss is very fast, and cell holds very much during longer-term storage
The problems such as easy apoptosis, the invention provides a kind of placenta and umbilical cord cells protection liquid.
Concrete technical scheme of the present invention is as follows：
The invention provides a kind of placenta and umbilical cord cells protection liquid, the protection liquid mainly by mycoplasma inhibitor and
Aminoglycoside antibiotics is formed with MEM-α culture medium aqueous dissolutions, and the MEM-α culture medium aqueous solution described in per mL dissolves respectively
The mycoplasma inhibitor 10-30mg, the aminoglycoside antibiotics 180-240U, the MEM-α culture medium aqueous solution
Concentration is 10-15mg/mL.
The protection liquid that the present invention is provided in the MEM-α culture medium aqueous solution by adding mycoplasma inhibitor and aminoglycoside
Class antibiotic, is that placenta stem-cell and umbilical cord mesenchymal stem cells provide suitable Conservation environment, by lot of experiment validation,
Two kinds of composition collaboration compatibilities are dissolved in the protection liquid energy constituted in the MEM-α culture medium aqueous solution and enough simulate human body environment, more fit
Suitable placenta stem-cell and umbilical cord mesenchymal stem cells are preserved, and are played a part of protecting cytoactive in transportation, are extended
Transport, storage time, prevent after cells ex vivo the Apoptosis in transport and storing process, are to deposit in placenta or umbilical cord cells later stage
Storage provides effectively basis.
It is auxiliary that every liter of MEM-α culture mediums aqueous solution that the present invention is provided includes polyvinylpyrrolidone 100g, reduced form
Enzyme II 5g, DMEM fluid nutrient mediums 500ml, three (methylol) methylglycine 25g, dexamethasone 100mg, Coomassie brilliant blue
R-250 5g, 3- [3- (courage amidopropyl) dimethylamino] propane sulfonic acid salt 5g, trypan blue 25g, N- lauryl creatine acid
Sodium 100G, sephadex G -25 [in] 25g, pepsin [1:3000] 25g, hyaluronidase 100mg, G-418 100mg,
DMEM/F12(1:1) fluid nutrient medium 500ml, isopropyl-β-D-thiogalactoside 1g, embedding medium 118ml, one-step method are total
RNA extracts reagents 200ml, cephalothin 100mg, Sodium Pyruvate 10g, spermine 1g, spermidine [essence is narrowed] 1g, Australia hyclone
500ml, hyclone 200ml, Proteinase K 100mg, Valine 25g, L-Leu 100g, L-Trp 10g, vitamin
C 25g, thymidine 5g, GSH 5g, adenosine -5'- triphosphoric acid disodiums 1g, ampicillin 1g, 3,3'- diamino
Base benzidine 10g, N- (2- ethoxys) piperazine-N'-2- ethane sulfonic acids 25g.
DMEM (Dulbecco ' s modified eagle medisum) fluid nutrient medium described above is to be a kind of containing each
Plant the culture medium of amino acid and glucose.Each composition and content are as follows in every liter of culture medium：Anhydrous calcium chloride 265mg/L, nine
Water ferric nitrate 0.1mg/L, potassium chloride 400mg/L, anhydrous magnesium sulfate 97.6mg/L 7, sodium chloride 6400mg/L, anhydrous phosphoric acid two
Hydrogen sodium 109mg/L, succinic acid 75mg/L, sodium succinate 100mg/L, L- R-gene 84mg/L, L- hydrochloric acid cystine 63mg/
L, glycine 30mg/L, L- histidine monohydrochloride 42mg/L, ILE 105mg/L, Serine 42mg/L, L-threonine
95mg/L, L-Trp 16mg/L, TYR 72mg/L, Valine 94mg/L, D-VB5 calcium 4mg/L, choline tartrate
7.2mg/L, folic acid 4mg/L, inositol 7.2mg/L, niacinamide 4mg/L, riboflavin 0.4mg/L, thiamine hydrochloride 4mg/L, hydrochloric acid pyrrole
Tremble pungent 4mg/L.Further to improve, DMEM culture mediums are low-sugar type DMEM culture mediums.
Further, the aminoglycoside antibiotics is in gentamicin, kanamycins, amikacin or TOB
One or more, it is preferred that the aminoglycoside antibiotics is by gentamicin and kanamycins according to 5:2 parts by weight
Than composition.
Further, the mycoplasma inhibitor includes the component of following parts by weight：20-40 parts of kitasamycin, rely
10-30 parts of ammonia tetracycline, 20-50 parts of gemifloxacin.Mycoplasma inhibitor includes kitasamycin, Armyl and Jimmy
Husky star can effectively suppress and kill the mycoplasma in protection liquid, and the action period is short, does not influence to fill between placenta stem-cell and umbilical cord
The preservation and metabolism of matter stem cell, and cell surface after treatment is smooth, and placenta stem-cell and umbilical cord mesenchyma are dry thin
Born of the same parents will not be re-infected by mycoplasma easily in transport and later stage storing process, and the existence of safety is provided for the storage of cell
Environment, the transport for placenta or umbilical cord provides protective effect.
Further, the mycoplasma inhibitor also includes the component of following parts by weight：5-10 parts of Rhizoma Curcumae extract, fish
4-8 parts of extract of raw meat grass, 3-6 parts of Rhizoma Smilacis Glabrae extract.Be stored with very big threat of the mycoplasma to cell is acted on, it is easy to made
Into cell infection, so that Apoptosis, is carried therefore, being further provided in the present invention and adding curcuma zedoary in mycoplasma inhibitor
Thing, houttuynia extract and Rhizoma Smilacis Glabrae extract are taken, these three traditional Chinese medicine ingredients can pass through on the basis of original western medicine composition
The effective component composition mycoplasma inhibitor of the combination of Chinese tradiational and Western medicine, effectively suppresses and kills the mycoplasma in protection liquid, action period
It is shorter, there is stronger protective effect to cell, cell infection is prevented.
Further, the protection liquid also contains HES and chitin, the MEM-α culture medium aqueous solution described in per mL
The HES 8-10mg, the chitin 1-5mg are dissolved respectively.By increasing HES and crust in the present invention
Matter can effectively maintain the osmotic equilibrium of placenta stem-cell and umbilical cord mesenchymal stem cells, play the work for the osmotic pressure for maintaining solution
With improving stability of solution, prevent Apoptosis, effectively simulate human body environment, a voltage stabilizing, stably, suitably is provided for cell
Living environment, extend the holding time of cell.
Further, the protection liquid also contains dextran and liquaemin, the MEM-α culture mediums aqueous solution point described in per mL
The dextran 4-6mg, the liquaemin 1-3mg are not dissolved.The dextran and liquaemin provided in the present invention can be made
For cryoprotective agent, the stability of solution is improved, a living environment being closer to human body is provided for cell, cell is reduced
The death rate, extends the holding time of cell.
Further, the protection liquid also contains sodium selenite and N-acetylcystein, MEM-α culture mediums described in per mL
The aqueous solution dissolves the sodium selenite 0.4-0.6mg, the N-acetylcystein 0.1-0.3mg respectively.What the present invention was provided
Sodium selenite and N-acetylcystein are added in protection liquid and can eliminate to preserve can cause cytoactive to reduce or death
Cytotoxin.Wherein, sodium selenite can promote hydroperoxides to be metabolized, and eliminate oxide enzyme and oxygen radical pair in protection liquid
The injury of cell, has good injury protection effect to stem cell on this point.In addition, N-acetylcystein is active oxygen
The scavenger of free radical, it can improve the damage of variety classes cell caused by oxidative stress；To being slowed down for sodium selenite
Outside other oxidative damages etc. made up, it is ensured that the gentle Conservation environment of cell.
Further, the protection liquid also contains lactobionic acid, blood albumin, sodium gluconate and gucosamine, per mL
MEM-α culture mediums the aqueous solution dissolves the lactobionic acid 5-8mg, the blood albumin 2-4mg, the gluconic acid respectively
Sodium 5-8mg, the gucosamine 4-6mg.Lactobionic acid, blood albumin, glucose are provided in the protection liquid provided in the present invention
Sour sodium, gucosamine, can provide certain nutritional ingredient for cell, while cellular immunity can be improved, prevent cell sense
Dye, suppresses Apoptosis, improves the survival rate of cell.
It is preferred that, the protection liquid is also containing cefoperazone sodium and phenol red, MEM-α culture mediums aqueous solution difference described in per mL
Dissolve the cefoperazone sodium 1-4mg, the phenol red 1-3mg.In the present invention by protection liquid in add cefoperazone sodium and
Phenol red effect is to be prevented and pointed out the pollution having occurred and that to the pollution that may occur, can be with after solution is contaminated
Display color, protects the color of liquid to may determine that cell contamination degree by observing, effectively reduces Apoptosis.
Present invention also offers the preparation method that a kind of placenta and umbilical cord cells protect liquid, the preparation method includes following step
Suddenly:
S1, take concentration be 10-15mg/mL the MEM- α culture medium aqueous solution, into the MEM- α culture medium aqueous solution plus
Enter aminoglycoside antibiotics, and be stirred, aminoglycoside antibiotics is substantially dissolved in the MEM- α culture medium aqueous solution
In, the content that the aminoglycoside antibiotics is dissolved in every mL MEM- α culture medium aqueous solution is 180-240U；
S2, it is dissolved with into step S1 in the MEM- α culture medium aqueous solution of aminoglycoside antibiotics and adds mycoplasma suppression
Preparation, and be stirred, mycoplasma inhibitor is substantially dissolved in the MEM- α culture medium aqueous solution, that is, obtain placenta and umbilical cord
The content that the mycoplasma inhibitor is dissolved in Cell protective solutions, every mL MEM- α culture medium aqueous solution is 10-30mg.
It is of the invention with existing patent provide protection liquid preparation method compared with, prepare it is more convenient, without specific ring
Border, can produce preparation in enormous quantities, not be affected by the external environment.
Beneficial effects of the present invention are as follows：The protection liquid that the present invention is provided can not only effectively kill or suppress mycoplasma
Formed, prevent placenta stem-cell and umbilical cord mesenchymal stem cells from infecting, and can be dry thin for placenta by adding protective ingredient
Born of the same parents and umbilical cord mesenchymal stem cells provide a stable osmotic pressure environment, effectively simulate human body environment, improve cells survival rate,
Nutritional ingredient can be provided for storage and transport in the cell short time simultaneously, improve the immune performance of cell, prevent cell infection,
The viability of cell is effectively improved, extends cell haulage time.
Embodiment
The present invention is described in further detail with reference to following examples.
Embodiment 1
The embodiment of the present invention 1 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor and gentamicin are formed with MEM-α culture medium aqueous dissolutions, and the MEM-α culture medium aqueous solution described in per mL dissolves respectively
The mycoplasma inhibitor 10mg, the gentamicin 180U, the concentration of the MEM-α culture medium aqueous solution is 10mg/mL.
Embodiment 2
The embodiment of the present invention 2 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin and kanamycins are formed with MEM-α culture medium aqueous dissolutions, and MEM-α culture mediums described in per mL are water-soluble
Liquid dissolves the mycoplasma inhibitor 20mg, the gentamicin 150U, the kanamycins 60U, the MEM-α cultures respectively
The concentration of the base aqueous solution is 12.5mg/mL.
Embodiment 3
The embodiment of the present invention 3 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, amikacin and TOB are formed with MEM-α culture medium aqueous dissolutions, MEM-α trainings described in per mL
Support the base aqueous solution dissolve the mycoplasma inhibitor 30mg respectively, it is the gentamicin 80U, the amikacin 80U, described appropriate
Obramycin 80U, the concentration of the MEM-α culture medium aqueous solution is 15mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：20 parts of kitasamycin, 10 parts of Armyl,
20 parts of gemifloxacin.
Present invention also offers the preparation method that a kind of placenta and umbilical cord cells protect liquid, the preparation method includes following step
Suddenly:
S1, take concentration be 15mg/mL the MEM- α culture medium aqueous solution, into the MEM- α culture medium aqueous solution add celebrating
Big mycin, amikacin and TOB, and be stirred, it is substantially dissolved in gentamicin, amikacin and TOB
In the MEM- α culture medium aqueous solution, the gentamicin 80U, the amikacin are dissolved in every mL MEM- α culture medium aqueous solution
80U, the TOB 80U；
S2, be dissolved with into step S1 in the MEM- α culture medium aqueous solution of gentamicin, amikacin and TOB plus
Enter mycoplasma inhibitor, and be stirred, mycoplasma inhibitor is substantially dissolved in the MEM- α culture medium aqueous solution, that is, obtain
Placenta and umbilical cord cells protect liquid, and the content that the mycoplasma inhibitor is often dissolved in the mL MEM- α culture medium aqueous solution is
30mg。
The compound method of mycoplasma inhibitor is：By 20 parts of kitasamycin, 10 parts of Armyl, 20 parts of gemifloxacin
Directly mix.
Embodiment 4
The embodiment of the present invention 4 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, kanamycins and amikacin are formed with MEM-α culture medium aqueous dissolutions, MEM-α trainings described in per mL
Support the base aqueous solution and dissolve the mycoplasma inhibitor 20mg, the gentamicin 70U, the kanamycins 70U and described respectively
Amikacin 70U, the concentration of the MEM-α culture medium aqueous solution is 11mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：30 parts of kitasamycin, 20 parts of Armyl,
35 parts of gemifloxacin.
The preparation method of the protection liquid provided in the present embodiment 4 is identical with the method for embodiment 3.
The compound method of mycoplasma inhibitor is：By 30 parts of kitasamycin, 20 parts of Armyl, 35 parts of gemifloxacin
Directly mix.
Embodiment 5
The embodiment of the present invention 5 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, kanamycins, amikacin and TOB are formed with MEM-α culture medium aqueous dissolutions, per mL institutes
State the MEM-α culture mediums aqueous solution and dissolve the mycoplasma inhibitor 10mg, the gentamicin 60U, the kanamycins respectively
60U, the amikacin 30U, the TOB 30U, the concentration of the MEM-α culture medium aqueous solution is 12mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：40 parts of kitasamycin, 30 parts of Armyl,
50 parts of gemifloxacin, 5 parts of Rhizoma Curcumae extract, 4 parts of houttuynia extract, 3 parts of Rhizoma Smilacis Glabrae extract.
The preparation method of the protection liquid provided in the present embodiment 5 is identical with the method for embodiment 3.
The compound method of mycoplasma inhibitor is：By 40 parts of kitasamycin, 30 parts of Armyl, gemifloxacin 50
Part, 5 parts of Rhizoma Curcumae extract, 4 parts of houttuynia extract, 3 parts of Rhizoma Smilacis Glabrae extract are directly mixed.
Embodiment 6
The embodiment of the present invention 6 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, kanamycins, amikacin and TOB are formed with MEM-α culture medium aqueous dissolutions, per mL institutes
State the MEM-α culture mediums aqueous solution and dissolve the mycoplasma inhibitor 10mg, the gentamicin 60U, the kanamycins respectively
60U, the amikacin 30U, the TOB 30U, the concentration of the MEM-α culture medium aqueous solution is 12mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：30 parts of kitasamycin, 20 parts of Armyl,
35 parts of gemifloxacin, 8 parts of Rhizoma Curcumae extract, 6 parts of houttuynia extract, 5 parts of Rhizoma Smilacis Glabrae extract.
The preparation method of the protection liquid provided in the present embodiment 6 is identical with the method for embodiment 3.
The compound method of mycoplasma inhibitor is：By 30 parts of kitasamycin, 20 parts of Armyl, gemifloxacin 35
Part, 8 parts of Rhizoma Curcumae extract, 6 parts of houttuynia extract, 5 parts of Rhizoma Smilacis Glabrae extract are directly mixed.
Embodiment 7
The embodiment of the present invention 7 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, HES and chitin are formed with MEM-α culture medium aqueous dissolutions, MEM-α trainings described in per mL
Support the base aqueous solution dissolve respectively the mycoplasma inhibitor 10mg, the gentamicin 180U, the HES 10mg and
The chitin 1mg, the concentration of the MEM-α culture medium aqueous solution is 10mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：20 parts of kitasamycin, 10 parts of Armyl,
20 parts of gemifloxacin, 10 parts of Rhizoma Curcumae extract, 8 parts of houttuynia extract, 6 parts of Rhizoma Smilacis Glabrae extract.
The present embodiment 7 provide protection liquid preparation method be by mycoplasma inhibitor 10mg, the gentamicin 180U,
The HES 10mg and the chitin 1mg are dissolved in the MEM-α culture medium aqueous solution respectively.
The compound method of mycoplasma inhibitor is：By 20 parts of kitasamycin, 10 parts of Armyl, gemifloxacin 20
Part, 10 parts of Rhizoma Curcumae extract, 8 parts of houttuynia extract, 6 parts of Rhizoma Smilacis Glabrae extract are directly mixed.
Embodiment 8
The embodiment of the present invention 8 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, HES and chitin are formed with MEM-α culture medium aqueous dissolutions, MEM-α trainings described in per mL
Support the base aqueous solution and dissolve the mycoplasma inhibitor 10mg, the gentamicin 180U, the HES 8mg and institute respectively
Chitin 3mg is stated, the concentration of the MEM-α culture medium aqueous solution is 10mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：40 parts of kitasamycin, 30 parts of Armyl,
50 parts of gemifloxacin, 5 parts of Rhizoma Curcumae extract, 4 parts of houttuynia extract, 3 parts of Rhizoma Smilacis Glabrae extract.
The present embodiment 8 provide protection liquid preparation method be by mycoplasma inhibitor 10mg, the gentamicin 180U,
The HES 8mg and the chitin 3mg are dissolved in the MEM-α culture medium aqueous solution respectively.
The compound method of mycoplasma inhibitor is：By 40 parts of kitasamycin, 30 parts of Armyl, gemifloxacin 50
Part, 5 parts of Rhizoma Curcumae extract, 4 parts of houttuynia extract, 3 parts of Rhizoma Smilacis Glabrae extract are directly mixed.
Embodiment 9
The embodiment of the present invention 9 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, HES, chitin, dextran and liquaemin are with MEM-α culture mediums aqueous dissolutions
Into the MEM-α culture mediums aqueous solution described in per mL dissolves the mycoplasma inhibitor 20mg, the gentamicin 210U, institute respectively
State HES 9mg, the chitin 5mg, the dextran 4mg, the liquaemin 1mg, the MEM-α culture medium water
The concentration of solution is 10mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：30 parts of kitasamycin, 20 parts of Armyl,
35 parts of gemifloxacin, 8 parts of Rhizoma Curcumae extract, 6 parts of houttuynia extract, 5 parts of Rhizoma Smilacis Glabrae extract.
The preparation method for the protection liquid that the present embodiment 9 is provided is by mycoplasma inhibitor 20mg, gentamicin 210U, hydroxyl second
Base starch 9mg, chitin 5mg, dextran 4mg, liquaemin 1mg are dissolved in the MEM-α culture medium aqueous solution respectively.
The compound method of mycoplasma inhibitor is：By 30 parts of kitasamycin, 20 parts of Armyl, gemifloxacin 35
Part, 8 parts of Rhizoma Curcumae extract, 6 parts of houttuynia extract, 5 parts of Rhizoma Smilacis Glabrae extract are directly mixed.
Embodiment 10
The embodiment of the present invention 10 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, HES, chitin, dextran, liquaemin, sodium selenite and N-acetylcystein are used
MEM-α culture medium aqueous dissolutions are formed, and the MEM-α culture mediums aqueous solution described in per mL dissolves the mycoplasma inhibitor respectively
10mg, the gentamicin 180U, the HES 8mg, the chitin 3mg, the dextran 6mg, the liver
Plain sodium 3mg, the sodium selenite 0.4mg, the N-acetylcystein 0.1mg, the concentration of the MEM-α culture medium aqueous solution
For 10mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：40 parts of kitasamycin, 30 parts of Armyl,
50 parts of gemifloxacin, 5 parts of Rhizoma Curcumae extract, 4 parts of houttuynia extract, 3 parts of Rhizoma Smilacis Glabrae extract.
The preparation method for the protection liquid that the present embodiment 10 is provided is by mycoplasma inhibitor 10mg, gentamicin 180U, hydroxyl
Hydroxyethyl starch 8mg, chitin 3mg, dextran 6mg, liquaemin 3mg, sodium selenite 0.4mg, N-acetylcystein 0.1mg
It is dissolved in respectively in the MEM-α culture medium aqueous solution.
The compound method of mycoplasma inhibitor is：By 40 parts of kitasamycin, 30 parts of Armyl, gemifloxacin 50
Part, 5 parts of Rhizoma Curcumae extract, 4 parts of houttuynia extract, 3 parts of Rhizoma Smilacis Glabrae extract are directly mixed.
Embodiment 11
The embodiment of the present invention 11 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, HES, chitin, dextran, liquaemin, sodium selenite, N-acetylcystein, breast
Uronic acid, blood albumin, sodium gluconate and gucosamine are formed with MEM-α culture medium aqueous dissolutions, MEM-α described in per mL
The culture medium aqueous solution dissolve respectively the mycoplasma inhibitor 20mg, the gentamicin 210U, the HES 9mg,
The chitin 1mg, the dextran 4mg, the liquaemin 1mg, the sodium selenite 0.6mg, the Guang of N- acetyl half
Propylhomoserin 0.3mg, the lactobionic acid 5mg, the blood albumin 2mg, the sodium gluconate 5mg, the gucosamine 4mg,
The concentration of the MEM-α culture medium aqueous solution is 10mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：30 parts of kitasamycin, 20 parts of Armyl,
35 parts of gemifloxacin, 8 parts of Rhizoma Curcumae extract, 6 parts of houttuynia extract, 5 parts of Rhizoma Smilacis Glabrae extract.
The preparation method for the protection liquid that the present embodiment 11 is provided is by mycoplasma inhibitor 20mg, gentamicin 210U, hydroxyl
Hydroxyethyl starch 9mg, chitin 1mg, dextran 4mg, liquaemin 1mg, sodium selenite 0.6mg, N-acetylcystein
0.3mg, lactobionic acid 5mg, blood albumin 2mg, sodium gluconate 5mg, gucosamine 4mg are dissolved in MEM-α culture mediums respectively
In the aqueous solution.
The compound method of mycoplasma inhibitor is：By 30 parts of kitasamycin, 20 parts of Armyl, gemifloxacin 35
Part, 8 parts of Rhizoma Curcumae extract, 6 parts of houttuynia extract, 5 parts of Rhizoma Smilacis Glabrae extract are directly mixed.
Embodiment 12
The embodiment of the present invention 12 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, HES, chitin, dextran, liquaemin, sodium selenite, N-acetylcystein, breast
Uronic acid, blood albumin, sodium gluconate, gucosamine, cefoperazone sodium and phenol red use MEM-α culture mediums aqueous dissolutions and
Into the MEM-α culture mediums aqueous solution described in per mL dissolves the mycoplasma inhibitor 10mg, the gentamicin 180U, institute respectively
State HES 8mg, the chitin 3mg, the dextran 6mg, the liquaemin 3mg, the sodium selenite
0.4mg, the N-acetylcystein 0.1mg, the lactobionic acid 8mg, the blood albumin 4mg, the sodium gluconate
8mg, the gucosamine 6mg, the cefoperazone sodium 1mg, the phenol red 1mg, the concentration of the MEM-α culture medium aqueous solution
For 10mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：40 parts of kitasamycin, 30 parts of Armyl,
50 parts of gemifloxacin, 5 parts of Rhizoma Curcumae extract, 4 parts of houttuynia extract, 3 parts of Rhizoma Smilacis Glabrae extract.
The preparation method for the protection liquid that the present embodiment 12 is provided is by mycoplasma inhibitor 10mg, gentamicin 180U, hydroxyl
Hydroxyethyl starch 8mg, chitin 3mg, dextran 6mg, liquaemin 3mg, sodium selenite 0.4mg, N-acetylcystein
0.1mg, lactobionic acid 8mg, blood albumin 4mg, sodium gluconate 8mg, gucosamine 6mg, cefoperazone sodium 1mg, phenol red 1mg
It is dissolved in respectively in the MEM-α culture medium aqueous solution.
The compound method of mycoplasma inhibitor is：By 40 parts of kitasamycin, 30 parts of Armyl, gemifloxacin 50
Part, 5 parts of Rhizoma Curcumae extract, 4 parts of houttuynia extract, 3 parts of Rhizoma Smilacis Glabrae extract are directly mixed.
Embodiment 13
The embodiment of the present invention 11 provides a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, HES, chitin, dextran, liquaemin, sodium selenite, N-acetylcystein, breast
Uronic acid, blood albumin, sodium gluconate, gucosamine, cefoperazone sodium and phenol red use MEM-α culture mediums aqueous dissolutions and
Into the MEM-α culture mediums aqueous solution described in per mL dissolves the mycoplasma inhibitor 20mg, the gentamicin 210U, institute respectively
State HES 9mg, the chitin 4mg, the dextran 4mg, the liquaemin 1mg, the sodium selenite
0.6mg, the N-acetylcystein 0.3mg, the lactobionic acid 5mg, the blood albumin 2mg, the sodium gluconate
5mg, the gucosamine 4mg, the cefoperazone sodium 4mg, the phenol red 3mg, the concentration of the MEM-α culture medium aqueous solution
For 10mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：30 parts of kitasamycin, 20 parts of Armyl,
35 parts of gemifloxacin, 8 parts of Rhizoma Curcumae extract, 6 parts of houttuynia extract, 5 parts of Rhizoma Smilacis Glabrae extract.
The preparation method for the protection liquid that the present embodiment 13 is provided is by mycoplasma inhibitor 20mg, gentamicin 210U, hydroxyl
Hydroxyethyl starch 9mg, chitin 4mg, dextran 4mg, liquaemin 1mg, sodium selenite 0.6mg, N-acetylcystein
0.3mg, lactobionic acid 5mg, blood albumin 2mg, sodium gluconate 5mg, gucosamine 4mg, cefoperazone sodium 4mg, phenol red 3mg
It is dissolved in respectively in the MEM-α culture medium aqueous solution.
The compound method of mycoplasma inhibitor is：By 30 parts of kitasamycin, 20 parts of Armyl, gemifloxacin 35
Part, 8 parts of Rhizoma Curcumae extract, 6 parts of houttuynia extract, 5 parts of Rhizoma Smilacis Glabrae extract are directly mixed.
Reference examples 1
Reference examples 1 of the present invention provide a kind of placenta and umbilical cord cells protection liquid, and the protection liquid will mainly be celebrated big mould
Element is formed with MEM-α culture medium aqueous dissolutions, and the MEM-α culture mediums aqueous solution described in per mL dissolves the gentamicin respectively
100U, the concentration of the MEM-α culture medium aqueous solution is 10mg/mL.
Reference examples 2
Reference examples 2 of the present invention provide a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor is formed with MEM-α culture medium aqueous dissolutions, and the MEM-α culture mediums aqueous solution described in per mL dissolves the mycoplasma respectively
Inhibitor 20mg, the concentration of the MEM-α culture medium aqueous solution is 10mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：15 parts of kitasamycin.
Reference examples 3
Reference examples 3 of the present invention provide a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor and gentamicin are formed with MEM-α culture medium aqueous dissolutions, and the MEM-α culture medium aqueous solution described in per mL dissolves respectively
The mycoplasma inhibitor 20mg, the gentamicin 180U, the concentration of the MEM-α culture medium aqueous solution is 10mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：30 parts of Armyl, 5 parts of Rhizoma Curcumae extract,
3 parts of Rhizoma Smilacis Glabrae extract.
Reference examples 4
Reference examples 4 of the present invention provide a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin and HES are formed with MEM-α culture medium aqueous dissolutions, MEM-α culture medium water described in per mL
Solution dissolves the mycoplasma inhibitor 20mg, the gentamicin 180U, the HES 10mg respectively, and the MEM-
The concentration of the α culture medium aqueous solution is 12.5mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：20 parts of kitasamycin, 10 parts of Armyl,
20 parts of gemifloxacin, 10 parts of Rhizoma Curcumae extract, 8 parts of houttuynia extract, 6 parts of Rhizoma Smilacis Glabrae extract.
Reference examples 5
Reference examples 5 of the present invention provide a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, HES, chitin and dextran are formed with MEM-α culture medium aqueous dissolutions, per mL institutes
State the MEM-α culture mediums aqueous solution and dissolve the mycoplasma inhibitor 20mg, gentamicin 180U, HES 9mg, first respectively
Chitin 1mg, dextran 4mg, the concentration of the MEM-α culture medium aqueous solution is 12.5mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：20 parts of kitasamycin, 10 parts of Armyl,
20 parts of gemifloxacin, 10 parts of Rhizoma Curcumae extract, 8 parts of houttuynia extract, 6 parts of Rhizoma Smilacis Glabrae extract.
Reference examples 6
Reference examples 6 of the present invention provide a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, HES, chitin, dextran, liquaemin and sodium selenite are water-soluble with MEM-α culture mediums
Liquid dissolving is formed, and the MEM-α culture mediums aqueous solution described in per mL dissolves the mycoplasma inhibitor 20mg, gentamicin respectively
180U, HES 9mg, chitin 4mg, dextran 4mg, liquaemin 3mg, sodium selenite 0.4mg, the MEM-α trainings
The concentration for supporting the base aqueous solution is 14mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：20 parts of kitasamycin, 10 parts of Armyl,
20 parts of gemifloxacin, 10 parts of Rhizoma Curcumae extract, 8 parts of houttuynia extract, 6 parts of Rhizoma Smilacis Glabrae extract.
Reference examples 7
Reference examples 6 of the present invention provide a kind of placenta and umbilical cord cells protection liquid, and the protection liquid is mainly by mycoplasma
Inhibitor, gentamicin, HES, chitin, dextran, liquaemin, sodium selenite, N-acetylcystein, breast
Uronic acid and blood albumin are formed with MEM-α culture medium aqueous dissolutions, and the MEM-α culture medium aqueous solution described in per mL dissolves respectively
The mycoplasma inhibitor 20mg, gentamicin 180U, HES 9mg, chitin 5mg, dextran 4mg, liquaemin
3mg, sodium selenite 0.4mg, N-acetylcystein 0.3mg, lactobionic acid 5mg, blood albumin 2mg, the MEM-α culture mediums
The concentration of the aqueous solution is 14mg/mL.
The mycoplasma inhibitor includes the component of following parts by weight：20 parts of kitasamycin, 10 parts of Armyl,
20 parts of gemifloxacin, 10 parts of Rhizoma Curcumae extract, 8 parts of houttuynia extract, 6 parts of Rhizoma Smilacis Glabrae extract.
Influence of the protection liquid that experiment 1, the present invention are provided to placenta stem-cell and umbilical cord mesenchymal stem cells survival rate
1st, sample is detected：
The protection liquid for taking the embodiment of the present invention 1, embodiment 2 and reference examples 1 to provide carries out detection experiment.
2nd, sample is gathered：
Contain 1.9 × 10 in 3 groups of cell masses of difference, first group of cell mass5Individual in vitro placenta stem-cell and umbilical cord mesenchyma
Contain 2.5 × 10 in stem cell, second group of cell mass5Individual in vitro placenta stem-cell and umbilical cord mesenchymal stem cells are the 3rd group thin
Contain 2.2 × 10 in born of the same parents group5Individual in vitro placenta stem-cell and umbilical cord mesenchymal stem cells, by first group, second group and the 3rd group
Cell mass is immersed in the protection liquid that embodiment 1, embodiment 2 and reference examples 1 are provided respectively, is protected under conditions of temperature is 4 DEG C
Deposit after 3 days, detect the quantity and growing state of placenta stem-cell and umbilical cord mesenchymal stem cells in different protection liquid.
| Group | Initial cell quantity | Cell quantity after preservation | Cell survival rate |
| Embodiment 1 | 1.9×105 | 1.69×105 | 89% |
| Embodiment 2 | 2.5×105 | 2.37×105 | 95% |
| Reference examples 1 | 2.2×105 | 1.4×105 | 64% |
Learnt by above-mentioned data, the protection liquid that the embodiment of the present invention 1, embodiment 2 are provided is compared with reference examples 1, the present invention
The protection liquid energy that embodiment 1, embodiment 2 are provided enough effectively improves cell survival rate, and in protection liquid mycoplasma inhibitor and
The content of gentamicin is higher, and the survival rate of its cell is higher, and the cell survival rate in the protection liquid that embodiment 2 is provided reaches
95%, therefore, by adding mycoplasma inhibitor in MEM-α culture medium solutions during the protection liquid that the present invention is provided can be verified
Human body environment can be effectively simulated with aminoglycoside antibiotics, cell survival rate is improved, Apoptosis, extension cell fortune is reduced
The defeated time, and the effect of the aminoglycoside antibiotics of the addition mixing of embodiment 2 is anti-significantly better than single aminoglycoside
Raw element.
The protection liquid that experiment 2, the present invention are provided is to placenta stem-cell and the survival rate test of umbilical cord mesenchymal stem cells
1st, sample is detected：There is provided using the protection liquid that embodiment 1,7,9,11 is provided as 1-4 groups, reference examples 1,4,5,7 are tested
Protection liquid control 1-4 groups.
2nd, sample is gathered：The umbilical cord cells or placenta cells of the equivalent of collection are submerged into above-mentioned experiment 1-4 groups and right respectively
According in 1-4 groups.
By the umbilical cord cells or placenta cells of each experimental group and control group respectively 24h, 48h, 96h, 240h, 480h,
960h and 1920h enter test procedure, and the density for adjusting umbilical cord cells or placenta cells is 1 × 106cells/mL.Press cell
Suspension：0.4% trypan blue=3:1(v:V) fully mix, take 20 μ L cell suspensions to add in cell counting count board, use Countstar
Cell counter carries out cytoactive rate detection.Cytoactive rate result is as shown in the table.
Experimental group and cellular control unit motility rate result
Note：" -- " is represented without activity.
From above-mentioned data, placenta stem-cell and umbilical cord mesenchymal stem cells that the protection liquid that embodiment 1 is provided is preserved
Cytoactive than using reference examples 1 provide protection liquid preserve placenta stem-cell and umbilical cord mesenchymal stem cells cell
Motility rate is high；The placenta stem-cell and the activity of umbilical cord mesenchymal stem cells that the protection liquid that embodiment 7,9,11 is provided respectively is preserved will
The placenta stem-cell and the Cell viability of umbilical cord mesenchymal stem cells preserved than the protection liquid provided using reference examples 1,4,5,7
It is high；And the placenta stem-cell and the time of umbilical cord mesenchymal stem cells that the protection liquid that embodiment 9 and 11 is provided is preserved will be compared
As usual 5 and 7 provide protection liquid preserve placenta stem-cell and umbilical cord mesenchymal stem cells time it is long, respectively reach 480h and
1920h。
It follows that lack in the protection liquid that the present invention is provided after mycoplasma inhibitor or aminoglycoside antibiotics, its
The motility rate of stem cell is significantly reduced.When the MEM-α culture medium solutions selected in protection liquid are addition HES and chitin
Afterwards, the motility rate of placenta stem-cell that protection liquid preserves and umbilical cord mesenchymal stem cells can be significantly improved, when HES and
Lack in the composition of chitin after one or composition substituted, lose the effect for improving stem cell motility rate；And in protection
The mixture of dextran and liquaemin is added in liquid can also improve protection liquid placenta stem-cell and umbilical cord mesenchymal stem cells
Holding time, the holding time can be brought up to 480h, when the composition of dextran and liquaemin lacks one or by other composition
After replacement, the holding time of placenta stem-cell and umbilical cord mesenchymal stem cells can shorten；Added in the protection liquid that the present invention is provided
Lactobionic acid, blood albumin, the mixture of sodium gluconate and gucosamine can also improve protection liquid placenta stem-cell and umbilical cord
The holding time of mescenchymal stem cell, the holding time can reach 1920h, and the motility rate of cell is still maintained at more than 65%.
Influence of the protection liquid that experiment 3, the present invention are provided to placenta stem-cell and umbilical cord mesenchymal stem cells immunity
1st, sample is detected：
The protection liquid for taking the embodiment of the present invention 3,5,10 and reference examples 2,3,6 to provide carries out detection experiment.
2nd, sample is gathered：
Contain 7.9 × 10 in 6 groups of cell masses of difference, first group of cell mass5Individual in vitro placenta stem-cell and umbilical cord mesenchyma
Contain 3.2 × 10 in stem cell, second group of cell mass5Individual in vitro placenta stem-cell and umbilical cord mesenchymal stem cells are the 3rd group thin
Contain 4.9 × 10 in born of the same parents group5In individual in vitro placenta stem-cell and umbilical cord mesenchymal stem cells, the 4th group of cell mass containing 3.5 ×
105Contain 4.2 × 10 in individual in vitro placenta stem-cell and umbilical cord mesenchymal stem cells, the 5th group of cell mass5Individual in vitro placenta is dry thin
Contain 3.2 × 10 in born of the same parents and umbilical cord mesenchymal stem cells, the 6th group of cell mass5Individual in vitro placenta stem-cell and umbilical cord mesenchyma are done
Cell, by first, second, third and fourth, five, six groups of cell masses be immersed in the guarantor that embodiment 3,5,10 and reference examples 2,3,6 are provided respectively
Protect in liquid, after being preserved 12 days under conditions of temperature is 4 DEG C, the placenta stem-cell and umbilical cord mesenchymal stem cells after culture are disappeared
Cell infection mycoplasma or the quantity of other bacteriums are detected with Flow cytometry after change processing, and it is former by branch to calculate cell
Body or infection；As a result such as following table：.
| Group | TCS amount | The cell quantity being uninfected by | Infection rate |
| Embodiment 3 | 7.9×105 | 5.7×105 | 28% |
| Embodiment 5 | 3.2×105 | 2.72×105 | 15% |
| Embodiment 10 | 4.9×105 | 4.61×105 | 6% |
| Reference examples 2 | 3.5×105 | 2.1×105 | 40% |
| Reference examples 3 | 4.2×105 | 3.15×105 | 25% |
| Reference examples 6 | 3.2×105 | 2.82×105 | 12% |
Drawn, contrasted from embodiment 3 and reference examples 2 by above-mentioned data, contained in the protection liquid that the present invention is provided
Gentamicin and mycoplasma inhibitor can effectively suppress the generation of bacterium or mycoplasma, while mycoplasma can be killed, confirm
Any one composition can not reach the effect for suppressing bacterium or mycoplasma.From embodiment 5 and reference examples 3, traditional Chinese and western medicine knot
The mycoplasma inhibitor being combined into can effectively reduce the formation of mycoplasma and bacterium, and mycoplasma suppression is disclosed in the present invention
Preparation can by kitasamycin, Armyl, gemifloxacin, Rhizoma Curcumae extract, houttuynia extract, Rhizoma Smilacis Glabrae extract
Relatively effective suppression and kill mycoplasma, from embodiment 10 and reference examples 6, the protection liquid energy that the present invention is provided is enough effective
The immunocompetence of cell is improved, cell infection mycoplasma is prevented.
Placenta or umbilical cord cells the protection liquid of the present invention is used to preserve after placenta or umbilical cord acquisition to umbilical cord or placenta point
From the umbilical cord or placenta of preceding people source.Placenta or umbilical cord the protection liquid of the present invention can be during placenta or umbilical cord be preserved
The metabolism of placenta or umbilical cord is reduced, the accumulation of metabolin is reduced, some basic nutriments and energy thing can be provided again
Matter, to maintain minimum metaboilic level, can effectively preserve the activity of the stem cell in placenta or umbilical cord, greatly reduce transport,
Handing-over, the time restriction of detection.The placenta or umbilical cord preserved through such a protection liquid, shadow of the Stem Cell Activity isolated by the time
Very little is rung, placenta or umbilical cord is greatly reduced from the time restriction for collecting preparation, and all the components used meet clinic
Standard, pollution rate is small.The placenta or umbilical cord preserved through this protection 2-10 DEG C of constant temperature of liquid, in placenta after preserving 48 hours or umbilical cord
Cytoactive is the 80% of the fresh human placenta or umbilical cord gathered, and the placenta or umbilical cord after collection preservation are preserved with any solution is not added with
Placenta or the pollution rate of umbilical cord compare, fall below 0.15% by 1.2%.
The present invention is not limited to above-mentioned preferred forms, and anyone can show that other are various under the enlightenment of the present invention
The product of form, however, make any change in its shape or structure, it is every that there is skill identical or similar to the present application
Art scheme, is within the scope of the present invention.
Claims (10)
1. a kind of placenta and umbilical cord cells protection liquid, it is characterised in that the protection liquid is mainly by mycoplasma inhibitor and ammonia
Base glycoside antibiotic is formed with MEM-α culture medium aqueous dissolutions, and the MEM-α culture mediums aqueous solution described in per mL dissolves institute respectively
State mycoplasma inhibitor 10-30mg, the aminoglycoside antibiotics 180-240U, the MEM-α culture medium aqueous solution it is dense
Spend for 10-15mg/mL.
2. placenta as claimed in claim 1 and umbilical cord cells protection liquid, it is characterised in that the aminoglycoside antibiotics is
One or more in gentamicin, kanamycins, amikacin or TOB.
3. placenta as claimed in claim 1 and umbilical cord cells protection liquid, it is characterised in that the mycoplasma inhibitor include with
The component of lower parts by weight：20-40 parts of kitasamycin, 10-30 parts of Armyl, 20-50 parts of gemifloxacin.
4. placenta as claimed in claim 3 and umbilical cord cells protection liquid, it is characterised in that the mycoplasma inhibitor also includes
The component of following parts by weight：5-10 parts of Rhizoma Curcumae extract, 4-8 parts of houttuynia extract, 3-6 parts of Rhizoma Smilacis Glabrae extract.
5. placenta as claimed in claim 1 and umbilical cord cells protection liquid, it is characterised in that the protection liquid also contains ethoxy
Starch and chitin, the MEM-α culture mediums aqueous solution described in per mL dissolve the HES 8-10mg, the chitin respectively
1-5mg。
6. placenta as claimed in claim 5 and umbilical cord cells protection liquid, it is characterised in that the protection liquid also contains dextrose
Acid anhydride and liquaemin, the MEM-α culture mediums aqueous solution described in per mL dissolve the dextran 4-6mg, the liquaemin 1- respectively
3mg。
7. placenta as claimed in claim 6 and umbilical cord cells protection liquid, it is characterised in that the protection liquid also contains selenous acid
Sodium and N-acetylcystein, the MEM-α culture mediums aqueous solution described in per mL dissolve the sodium selenite 0.4-0.6mg, institute respectively
State N-acetylcystein 0.1-0.3mg.
8. placenta as claimed in claim 7 and umbilical cord cells protection liquid, it is characterised in that the protection liquid also contains lactose aldehyde
Acid, blood albumin, sodium gluconate and gucosamine, the MEM-α culture mediums aqueous solution described in per mL dissolve the lactose aldehyde respectively
Sour 5-8mg, the blood albumin 2-4mg, the sodium gluconate 5-8mg, the gucosamine 4-6mg.
9. placenta as claimed in claim 1 and umbilical cord cells protection liquid, it is characterised in that the protection liquid also contains cephalo piperazine
Ketone sodium and phenol red, the MEM-α culture mediums aqueous solution described in per mL dissolves the cefoperazone sodium 1-4mg, the phenol red 1- respectively
3mg。
10. a kind of placenta and umbilical cord cells protect the preparation method of liquid, it is characterised in that the preparation method comprises the following steps:
S1, take concentration be 10-15mg/mL the MEM- α culture medium aqueous solution, add ammonia into the MEM- α culture medium aqueous solution
Base glycoside antibiotic, and be stirred, aminoglycoside antibiotics is substantially dissolved in the MEM- α culture medium aqueous solution, often
The content that the aminoglycoside antibiotics is dissolved in the mL MEM- α culture medium aqueous solution is 180-240U；
S2, addition mycoplasma inhibitor in the MEM- α culture medium aqueous solution of aminoglycoside antibiotics is dissolved with into step S1,
And be stirred, mycoplasma inhibitor is substantially dissolved in the MEM- α culture medium aqueous solution, that is, obtain placenta and umbilical cord cells are protected
Protect in liquid, every mL MEM- α culture medium aqueous solution and dissolve the content of the mycoplasma inhibitor for 10-30mg.
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