CN108130308A - A kind of umbilical cord tissue cryopreservation resuscitation method - Google Patents
A kind of umbilical cord tissue cryopreservation resuscitation method Download PDFInfo
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The present invention provides a kind of method of umbilical cord tissue cryopreservation resuscitation, comprising：Step S1 cuts umbilical cord tissue to obtain the first tissue item；Step S2, the first tissue item is immersed in vetrifying solution, then directly freezes the first tissue item to obtain minor microstructure item；Step S3 recovers the minor microstructure item to obtain third strip of tissue.Wherein, the vetrifying solution includes the first vetrifying solution, the second vetrifying solution and third vetrifying solution, the step S2 includes the first tissue item being immersed in successively in first vetrifying solution, second vetrifying solution and the third vetrifying solution, then the first tissue item is moved into freezen protective in liquid nitrogen, to obtain the minor microstructure item.The present invention is frozen and is recovered using Vitrification the umbilical cord tissue item, the necrosis rate of the umbilical cord tissue during reducing cryopreservation resuscitation while shortening the operating time and improving and freeze efficiency.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of umbilical cord tissue cryopreservation resuscitation method and mesenchyma are done carefully
Born of the same parents' separation method.
Background technology
Umbilical cord is the important substance of fetal period connection parent and fetus, is cord structures, and outer is one layer of amnion, is included
Two arteria umbilicalis and a umbilical vein, containing the special myxoid umbilical cord tissue of embryo between blood vessel and amnion, from umbilical cord group
Isolated mescenchymal stem cell can be used for treating a variety of diseases in knitting.
The common method for obtaining mescenchymal stem cell is first to isolate mescenchymal stem cell freezing from umbilical cord tissue to protect
It deposits, when use thaws processing again, this process time is long, and operation difficulty is big；And umbilical cord tissue is first preserved, it waits to need to use mesenchyma
It is recovered again to umbilical cord tissue during stem cell and separating treatment, then can save the time and reduce operation difficulty.But umbilical cord group
Being woven in vigor under ex vivo can decline gradually, and then influence the quality and quantity of isolated mescenchymal stem cell how
It is the current a great problem for hindering clinical practice that umbilical cord tissue can be preserved for a long time and maintain the activity of mescenchymal stem cell.
The Chinese invention patent of Publication No. CN104145943 discloses a kind of cryopreservation methods of umbilical cord tissue, the patent
With containing 5-10% permeability cryoprotector, 1-5% impermeabilities cryoprotector, serum replacement and basal medium
Frozen stock solution cools down to the umbilical cord tissue obtained after stripping blood vessel into line program, to preserve umbilical cord tissue.But when the above method operates
Between it is long and the necrosis rate of umbilical cord tissue is higher during freezing.
Therefore, it is necessary to a kind of novel umbilical cord tissue processing method is designed to solve above-mentioned technical problem.
Invention content
The present invention provides a kind of simple and effective umbilical cord tissue cryopreservation resuscitation methods, to obtain mescenchymal stem cell, keep away
The problem of exempting from operating time length of the existing technology and high umbilical cord tissue necrosis rate.
To achieve these goals, technical scheme of the present invention includes：
Step S1：Umbilical cord tissue is obtained, cuts the umbilical cord tissue to obtain the first tissue item；
Step S2：The first tissue item is immersed in vetrifying solution, then directly freeze the first tissue item with
Obtain minor microstructure item；
Step S3：The minor microstructure item recover to obtain third strip of tissue.
The vetrifying solution includes the first vetrifying solution, the second vetrifying solution and third vetrifying solution, the step S2 packets
It includes and the first tissue item is immersed in first vetrifying solution, second vetrifying solution and the third vitrifying successively
In liquid, the first tissue item is then moved into freezen protective in liquid nitrogen, obtains the minor microstructure item.
The cryopreservation resuscitation method of umbilical cord tissue of the present invention, advantage are：By the first tissue item successively
It is immersed in first vetrifying solution, second vetrifying solution and the third vetrifying solution, using the side directly frozen
Method freezes the first tissue item to obtain the minor microstructure item, shortens temperature fall time, can effectively prevent intracellular ice
Brilliant formation reduces tissue necrosis and apoptosis, and raising freezes and resuscitation effect.
Further, the umbilical cord tissue includes rete layer, and the step S1 includes tearing off the described of the umbilical cord tissue
Rete layer.
Further, the umbilical cord tissue includes arteria umbilicalis, and the step S1 includes tearing off the described of the umbilical cord tissue
Arteria umbilicalis.
Further, the first tissue item is in first vetrifying solution, second vetrifying solution and the third
Soaking time in vetrifying solution is 2-7min, first vetrifying solution, second vetrifying solution and the third glass
The temperature of glass liquid is 2-6 DEG C.
Further, the third vetrifying solution includes impermeability protective agent, permeability protective agent, thickener, KSR blood
Cleer and peaceful DMEM culture mediums, a concentration of 0.4-1.0mol/L of the impermeability protective agent, the protectant volume of permeability
Percentage is 10-30%, and the percent by volume of the thickener is 0.5-5%, and the percent by volume of the KSR serum is 10-
20%.
Further, the permeability protective agent includes dimethyl sulfoxide (DMSO) or ethylene glycol, the impermeability protective agent packet
Include any one or more in sucrose, glucose or trehalose, the thickener include chondroitin sulfate, dextran,
In Sodium Hyaluronate, carboxylic propyl methocel, carboxymethyl cellulose, polyvinylpyrrolidone, POLYPROPYLENE GLYCOL or polyvinyl alcohol
Any one or more.
Further, the component of second vetrifying solution presses volume percentage, including：The third vetrifying solution
40%-60%, the KSR serum substitutes 10-20%, surplus are the DMEM culture mediums.
Further, the component of first vetrifying solution presses volume percentage, including：The third vetrifying solution
20%-30%, the KSR serum substitutes 10-20%, surplus are the DMEM culture mediums.
Further, the step S3 includes handling the minor microstructure item with resuscitation fluid, and the resuscitation fluid includes first
Resuscitation fluid, the second resuscitation fluid and third resuscitation fluid, the processing procedure include the minor microstructure item being immersed in successively described
In first resuscitation fluid, second resuscitation fluid and the third resuscitation fluid.
Further, the minor microstructure item is recovered in first resuscitation fluid, second resuscitation fluid and the third
The immersion treatment time in liquid is 2-15min, first resuscitation fluid, second resuscitation fluid and the third resuscitation fluid
Temperature is 20-38 DEG C.
Further, first resuscitation fluid includes glucide, the KSR serum and the DMEM culture mediums, described
A concentration of 0.4-1.0mol/L of glucide, the percents by volume of the KSR serum substitutes are 10-20%, the carbohydrate
Substance includes any one or more in sucrose, trehalose or glucose；The component of second resuscitation fluid presses volume basis
Than counting, including：The first resuscitation fluid 40%-60%, the KSR serum substitutes 10-20%, surplus are DMEM culture mediums；
The component of the third resuscitation fluid presses volume percentage, including：KSR serum substitute 10-20%, surplus are cultivated for DMEM
Base.
Further, the step S3 includes the third strip of tissue described in culture solution culture.
Further, the culture solution includes vitamin, tert-butyl hydroquinone, Caspase inhibition
Agent, N-acetyl-L-cysteine, the DMEM culture mediums and the KSR serum substitutes.
Another aspect of the present invention is to provide a kind of separation method of mescenchymal stem cell, includes the following steps：It obtains
The third strip of tissue obtained using claim 1-13 any one of them umbilical cord tissue cryopreservation resuscitations method, to the third group
It knits item and carries out culture and digestion process successively to form cell suspension and fragment of tissue, remove described in the fragment of tissue and culture
Cell suspension is to form the mescenchymal stem cell.
Further, the tissue pieces carry out the digestion process with tissue digestion liquid, and the tissue digestion liquid is glue
The mixture of protoenzyme, dispase and hyaluronidase.
Term：
As used herein, unless otherwise stated, " Caspase inhibitors " refer to
Caspase inhibitor, Caspase is one group and is present in protease with similar structure in cytoplasm, with eukaryocyte apoptosis
It is closely related, and the growth, differentiation and apoptosis that participate in cell are adjusted.Caspase inhibitor plays effectively different Caspase
Inhibiting effect, select different Caspase inhibitor that can play the role of inhibiting Apoptosis in different situations, prompting faces
It can be used for the treatment of a variety of apoptosis-associated diseases on bed.
Beneficial effects of the present invention are as follows：
(1) present invention uses directly to freeze and the strip of tissue is frozen and recovered with resuscitation technique, shortens cooling
Time can effectively prevent the formation of intracellular ice crystal, reduce tissue necrosis and apoptosis, and raising freezes and resuscitation effect.
(2) present invention freeze processing before first by the amnion of umbilical cord tissue outer layer remove, be conducive to the first vetrifying solution,
It second vetrifying solution or the infiltration of third vetrifying solution and is evenly distributed in umbilical cord tissue, during effectively reducing cryopreservation resuscitation
Damage to tissue.
(3) first stripping removes the arteria umbilicalis of umbilical cord tissue before freezing and storing umbilical tissue of the present invention, retains umbilical vein, increases list
The umbilical cord mesenchymal stem cells of bit length freeze quantity, so as to improve cell yield.
(4) to resuscitation fluid, treated that minor microstructure item carries out culture processing, Neng Gouyou using the culture solution by the present invention
Effect increases the activity organized after recovery, improves the yield of mescenchymal stem cell.
Description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention, for those of ordinary skill in the art, without creative efforts, can be with
Other attached drawings are obtained according to these attached drawings.
Fig. 1 is the structural form figure of magnificent Tong Shi glue tissue that different disposal method obtains in the embodiment of the present invention；
Fig. 2 is to obtain cell quantity in suspension in the embodiment of the present invention after the different disposal group treatments of the sample of unit length；
Mescenchymal stem cell isolated for different disposal group sample in the embodiment of the present invention Fig. 3 is through identical incubation time
Expression activitiy after culture.
Specific embodiment
Unless otherwise defined, the technical term or scientific terminology used in claims of the present invention and specification should
The ordinary meaning understood by the personage in the technical field of the invention with general technical ability.
Purpose, technical scheme and advantage to make the embodiment of the present invention are clearer, below in conjunction with specific embodiment to this
Invention is described further, it should be pointed out that embodiment described below is intended to convenient for the understanding of the present invention, and to the present invention
Claimed range does not play any restriction effect.Based on the embodiments of the present invention, those of ordinary skill in the art are not having
All other embodiments obtained under the premise of creative work are made, shall fall within the protection scope of the present invention.
Method therefor is conventional method unless otherwise specified in following embodiments, and agents useful for same commercially obtains
.
Reagent：
KSR serum substitutes are produced for ThermoFisher companies, and product identification is：10828028；
DMEM culture mediums are produced for ThermoFisher companies, and product identification is：11885092；
Dimethyl sulfoxide (DMSO) is produced for Sigma-Aldrich companies, and product identification is：34869；
Ethylene glycol is produced for Sigma-Aldrich companies, and product identification is：293237；
Polyvinylpyrrolidone is produced for Sigma-Aldrich companies, and product identification is：P0930；
Glucose is produced for Sigma-Aldrich companies, and product identification is：V900392；
Sucrose is produced for Sigma-Aldrich companies, and product identification is：V900116；
Trehalose is produced for Sigma-Aldrich companies, and product identification is：T0167.
Of the existing technology to solve the problems, such as, an embodiment of the present invention provides a kind of cryopreservation resuscitation sides of umbilical cord tissue
Method, including：
Step S1：Umbilical cord tissue is obtained, cuts the umbilical cord tissue to obtain the first tissue item；
Step S2：The first tissue item is immersed in vetrifying solution, then directly freeze the first tissue item with
Obtain minor microstructure item；
Step S3：The minor microstructure item recover to obtain third strip of tissue.
The step S1 is specifically included：
Umbilical cord tissue is obtained, the umbilical cord tissue of acquisition will be cut into segment；
The length of the umbilical cord tissue after being cut in the present embodiment is 1 centimetre.After being cut in some embodiments of the invention
The umbilical cord tissue length be more than 1 centimetre be less than or equal to 2 centimetres.
Remove remaining blood or clot in the umbilical cord tissue after cutting；
The rete layer is torn off along the umbilical vein direction；
The present embodiment tears off method using passivity and tears off the rete layer.
First arteria umbilicalis and second navel are torn off along the trend of first arteria umbilicalis and second arteria umbilicalis
Artery retains the umbilical cord tissue for including the umbilical vein, and mesenchyma abundant the pipe Zhou Hanyou of the umbilical vein is done carefully
Born of the same parents；
Cutting includes the umbilical cord tissue of the umbilical vein, to form the first tissue item；
The width of the first tissue item described in the present embodiment is 2mm.The first tissue item described in some embodiments of the invention
Width be 1mm or 3mm.
The step S2 is specifically included：
Prepare the third vetrifying solution, second vetrifying solution and the first vetrifying solution；
The third vetrifying solution includes impermeability protective agent, permeability protective agent, thickener, KSR serum and DMEM
Culture medium.A concentration of 0.4-1.0mol/L of the impermeability protective agent, the protectant percent by volume of permeability are
10-30%, the percent by volume of the thickener is 0.5-5%, and the percent by volume of the KSR serum is 10-20%.
The permeability protective agent includes dimethyl sulfoxide (DMSO) or ethylene glycol, and the impermeability protective agent includes sucrose, Portugal
Grape sugar or trehalose in any one or more, the thickener include chondroitin sulfate, dextran, hyaluronic acid
It is any one in sodium, carboxylic propyl methocel, carboxymethyl cellulose, polyvinylpyrrolidone, POLYPROPYLENE GLYCOL or polyvinyl alcohol
Kind is a variety of.
Impermeability protective agent described in the present embodiment is sucrose, and the permeability protective agent is dimethyl sulfoxide (DMSO), described
Thickener is polyvinyl alcohol, and the sucrose concentration is 0.4mol/L, and the dimethyl sulfoxide (DMSO) percent by volume is 30%, described poly-
Vinyl alcohol percent by volume is 0.5%.Impermeability protective agent described in some embodiments of the invention be glucose or trehalose,
The permeability protective agent is ethylene glycol, and the thickener is chondroitin sulfate, dextran, Sodium Hyaluronate, carboxylic propyl first
Base cellulose, carboxymethyl cellulose, polyvinylpyrrolidone or POLYPROPYLENE GLYCOL, a concentration of 0.5mol/ of permeability protective agent
L, 0.6mol/L, 0.8mol/L or 1.0mol/L, the dimethyl sulfoxide (DMSO) percent by volume are 25%, 20%, 15% or 10%,
The thickening agent volume percentage is 1.0%, 2.0%, 3.0%, 4.0% or 5.0%.
The component of second vetrifying solution presses volume percentage, including：The third vetrifying solution 40%-60%,
The KSR serum substitutes 10-20%, surplus are the DMEM culture mediums.
The component of second vetrifying solution described in the present embodiment presses volume percentage, including：The third vetrifying solution
50%th, the KSR serum substitutes 20%.The component of second vetrifying solution described in some embodiments of the invention presses volume basis
Than counting, including：The third vetrifying solution 40% or 60%, the KSR serum substitutes 10% or 15%.
The component of first vetrifying solution presses volume percentage, including：The third vetrifying solution 20%-30%,
The KSR serum substitutes 10-20%, surplus are the DMEM culture mediums.
The component of first vetrifying solution described in the present embodiment presses volume percentage, including：The third vetrifying solution
25%th, the KSR serum substitutes 20%.The component of first vetrifying solution described in some embodiments of the invention presses volume basis
Than counting, including：The third vetrifying solution 20% or 30%, the KSR serum substitutes 10% or 15%.
The first tissue item is soaked in immersion treatment in the first vetrifying solution；
The first tissue item from the first vetrifying solution is taken out, is then transferred into the second vetrifying solution at immersion
Reason；
The first tissue item from the second vetrifying solution is taken out, is then transferred into third vetrifying solution at immersion
Reason；
The first tissue item is in first vetrifying solution, second vetrifying solution and the third vetrifying solution
Soaking time be 2-7min, the temperature of first vetrifying solution, second vetrifying solution and the third vetrifying solution
Degree is 2-6 DEG C.
In the present embodiment, the first tissue article is in first vetrifying solution, second vetrifying solution and described
Soaking time in three vetrifying solutions is 4min, first vetrifying solution, second vetrifying solution and the third glass
The temperature of glass liquid is 4 DEG C.The first tissue article described in some embodiments of the invention is in first vetrifying solution, described
Soaking time in two vetrifying solutions and the third vetrifying solution is 2min, 5min or 7min, first vetrifying solution,
The temperature of second vetrifying solution and the third vetrifying solution is 2 DEG C, 5 DEG C or 6 DEG C.
The first tissue item from the third vetrifying solution is taken out, is then transferred in liquid nitrogen rapidly, makes its temperature
Rapid drawdown obtains the minor microstructure item, and the minor microstructure item is preserved for a long time in liquid nitrogen.
The step S3 is specifically included：
Prepare first resuscitation fluid, the second resuscitation fluid and third resuscitation fluid；
First resuscitation fluid includes glucide, the KSR serum and the DMEM culture mediums, the glucide
A concentration of 0.4-1.0mol/L, the percent by volume of the KSR serum substitutes is 10-20%, and the glucide includes sugarcane
Any one or more in sugar, trehalose or glucose.
Glucide described in the present embodiment is sucrose, and the sucrose concentration is 0.4mol/L.Some implementations of the present invention
In example, the glucide is trehalose or glucose, the glucide a concentration of 0.6mol/L, 0.8mol/L or
1.0mol/L。
The component of second resuscitation fluid presses volume percentage, including：It is the first resuscitation fluid 40%-60%, described
KSR serum substitutes 10-20%, surplus are DMEM culture mediums.
The component of second resuscitation fluid described in the present embodiment presses volume percentage, including：First resuscitation fluid 50%,
The KSR serum substitutes 20%.The component of second resuscitation fluid described in some embodiments of the present invention presses volume percentage,
Including：First resuscitation fluid 40% or 60%, the KSR serum substitutes 10% or 15%.
The component of the third resuscitation fluid presses volume percentage, including：KSR serum substitute 10-20%, surplus are
DMEM culture mediums.
The component of third resuscitation fluid described in the present embodiment includes the KSR serum substitutes that percent by volume is 20%.This hair
Third resuscitation fluid component described in some bright embodiments includes the KSR serum substitutes that percent by volume is 10% or 15%.
The minor microstructure item is taken out from liquid nitrogen, it is put into rapidly immersion treatment in the first resuscitation fluid；
The minor microstructure item from first resuscitation fluid is taken out, is then moved into the second resuscitation fluid at immersion
Reason；
The minor microstructure item from second resuscitation fluid is taken out, is then moved into third resuscitation fluid at immersion
Reason；
Immersion of the minor microstructure item in first resuscitation fluid, second resuscitation fluid and the third resuscitation fluid
Processing time is 2-15min, and the temperature of first resuscitation fluid, second resuscitation fluid and the third resuscitation fluid is
20-38℃。
Minor microstructure item described in the present embodiment in first resuscitation fluid immersion treatment time be 3min, described first
The temperature of resuscitation fluid is 37 DEG C.In some embodiments of the present invention, the minor microstructure item impregnates in first resuscitation fluid
Processing time is 2min, 4min or 5min, and the temperature of first resuscitation fluid is 35 DEG C, 36 DEG C or 38 DEG C.
Minor microstructure item described in the present embodiment in second resuscitation fluid immersion treatment time be 5min, described second
The temperature of resuscitation fluid is 25 DEG C.In some embodiments of the present invention, the minor microstructure item impregnates in second resuscitation fluid
Processing time is 4min or 6min, and the temperature of second resuscitation fluid is 23 DEG C, 26 DEG C or 28 DEG C.
The immersion treatment time in the third resuscitation fluid of minor microstructure item described in the present embodiment is 10min, described the
The temperature of three resuscitation fluids is 25 DEG C.In some embodiments of the present invention, the minor microstructure item soaks in the third resuscitation fluid
It is 6min, 8min or 12min to steep processing time, and the temperature of the third resuscitation fluid is 23 DEG C, 26 DEG C or 28 DEG C.
The minor microstructure item from the third resuscitation fluid is taken out, then moves into the culture solution and is trained
It supports, obtains the third strip of tissue.
The culture solution include vitamin, tert-butyl hydroquinone, Caspase inhibitors, N- acetyl-
L-cysteine, the DMEM culture mediums and the KSR serum substitutes.
Vitamin described in the present embodiment is vitamin C, and the Caspase inhibitors are Z-
VAD-FMK, the percent by volume of the KSR serum substitutes is 20%.In some embodiments of the invention, the vitamin is dimension
Raw element E.
In some embodiments of the invention, the third strip of tissue is cut into size as 2mm × 2mm, 1mm × 1mm or 1mm
× 2mm fragments, with the culture solution culture.
The cryopreservation resuscitation method of the umbilical cord tissue provided in an embodiment of the present invention can effectively keep the umbilical cord tissue
Structure and form integrality, described in the present embodiment will be obtained by umbilical cord tissue cryopreservation resuscitation method described in the present embodiment
Third strip of tissue carries out form and knot with fresh strip of tissue and using the umbilical cord tissue that prior art cryopreservation resuscitation method obtains
Comparison on structure, to illustrate above-mentioned advantageous effect.
Specifically, the umbilical cord of same source is taken to be divided into three groups, is respectively processed, tissue sample is divided into A groups that treated,
B groups and C groups：
A groups are fresh strip of tissue, refer to the first tissue handled using the present embodiment step S1 the methods
Item.
B groups are the institute that is frozen using the cryopreservation resuscitation method of umbilical cord tissue described in the present embodiment and obtained after being recovered
State third strip of tissue；
C groups are frozen for the prior art, recover after umbilical cord tissue, answered with reference to freezing in patent US 2016/0066566A
Soviet side's method obtains.
The tissue sample of A groups, B groups and C groups using HE is dyed respectively, obtains the umbilical cord tissue that different disposal method obtains
Structural form figure, as shown in Figure 1, A be A groups umbilical cord tissue structure and form, B be B groups umbilical cord tissue structure with
Form, C are the structure and form of the umbilical cord tissue of C groups, and as seen in Figure 1, compared with A groups, the umbilical cord tissue of C groups is had time
Hole, institutional framework have shifting phenomena；And B groups are almost consistent with the structural form of A groups.The result shows that freezing using the present embodiment
The structure and form for the third strip of tissue that method for resuscitation obtains are complete, and this method can effectively reduce group during cryopreservation resuscitation
The necrosis knitted and apoptosis.
The cryopreservation resuscitation method of the umbilical cord tissue provided in an embodiment of the present invention, moreover it is possible to effectively improve the mesenchyma of acquisition
The quantity of stem cell simultaneously keeps its activity, and the present embodiment is to third tissue described in the cryopreservation resuscitation method using the umbilical cord tissue
Item detaches with fresh strip of tissue and using the identical enzymic digestion of the umbilical cord tissue carry out that prior art cryopreservation resuscitation method obtains
Processing, is compared the quantity of the mescenchymal stem cell of acquisition, to illustrate above-mentioned advantageous effect.
Specifically, the umbilical cord tissue of same source is taken to be divided into three parts to be respectively processed, tissue sample difference that treated
For D groups, E groups and F groups, specifically：
D groups are flesh tissue item, with reference to the preparation method of A groups in the present embodiment；
E groups are the third strip of tissue obtained using the cryopreservation resuscitation method of umbilical cord tissue described in the present embodiment；
F groups are to utilize the umbilical cord tissue of all blood vessels of reservation after prior art cryopreservation resuscitation method cryopreservation resuscitation, reference
Cryopreservation resuscitation method in patent US 2016/0066566A obtains.
The tissue sample obtained from D groups, E groups, F groups is subjected to mescenchymal stem cell separation with identical method.Separation side
Method is as follows：The strip of tissue that D groups, E groups, F groups obtain is cut into the tissue pieces of 2mm × 2mm, the obtained tissue pieces are used
Tissue digestion liquid digestion process is single cell suspension and a small amount of tissue pieces, and sampling counts, and obtains initial cell quantity, described
Tissue digestion liquid be clostridiopetidase A, the mixture of dispase, hyaluronidase, as a result see Fig. 2, D disappears for D group tissue samples through enzyme
The mescenchymal stem cell quantity obtained in suspension is obtained after change；E obtains what is obtained in suspension for E group tissue samples after enzymic digestion
Mescenchymal stem cell quantity；F obtains the mescenchymal stem cell quantity obtained in suspension for F group tissue samples after enzymic digestion.From
As it can be seen that the mescenchymal stem cell quantity that D groups obtain is 1 × 10 in Fig. 26A/cm；The mescenchymal stem cell quantity that E groups obtain is 6
×105A/cm；The mescenchymal stem cell quantity that F groups obtain is 1.5 × 106A/cm.The above result shows that removal arteria umbilicalis, guarantor
The D groups of umbilical vein are stayed compared with the F groups for retaining all blood vessels, retain umbilical vein D groups remain it is most of in umbilical cord tissue
Mescenchymal stem cell；E groups are compared with D groups, the results showed that are trained after being frozen, recover and recovered using the present embodiment the method
Processing is supported, can effectively obtain the mesenchyma liver cell in umbilical cord tissue and keeps its activity.
The embodiment of the present invention additionally provides a kind of separation method of mescenchymal stem cell, includes the following steps：Using above-mentioned
The third strip of tissue that the umbilical cord tissue cryopreservation resuscitation method obtains, cultivates it successively and digestion process is to form
Cell suspension and fragment of tissue remove the fragment of tissue and cultivate the cell suspension to form the mescenchymal stem cell.
The third strip of tissue carries out the digestion process with tissue digestion liquid, and the tissue digestion liquid is clostridiopetidase A, divides
Dissipate the mixture of enzyme and hyaluronidase.
The third strip of tissue is inoculated into culture bottle in the present embodiment, the culture bottle is placed in 37 DEG C, 5% titanium dioxide
It is cultivated 8-12 days in carbocyclic ring border.
After the cell suspension that the removal fragment of tissue obtains is continued culture 3-10 days in the present embodiment, with
Obtain mescenchymal stem cell.
The separation method of the mescenchymal stem cell provided in an embodiment of the present invention can be effectively increased the work organized after recovery
Property, improve the yield of mescenchymal stem cell, the present embodiment in same time, the mescenchymal stem cell that different samples are isolated
Culture activity be compared, to illustrate above-mentioned advantageous effect.
Specifically, the umbilical cord of same source is taken, be divided into three groups and is respectively processed, treated, and tissue sample divides
Not Wei G groups, H groups and I groups, specifically：
G groups are single cell suspension and a small amount of tissue pieces after the enzymic digestion obtained with reference to the preparation method of above-mentioned D groups；
H groups are single cell suspension and a small amount of tissue pieces after the enzymic digestion obtained with reference to the preparation method of above-mentioned E groups；
I groups are single cell suspension and a small amount of tissue pieces after the enzymic digestion obtained with reference to the preparation method of above-mentioned F groups.
By G groups, three groups of single cell suspensions of H groups and I groups and a small amount of tissue pieces centrifuge, discard supernatant, collect it is unicellular and
A small amount of tissue pieces, are inoculated into culture bottle respectively, and the culture bottle is placed in 37 DEG C, cultivates 8-12 in 5% carbon dioxide environment
My god, the fragment of tissue floated in culture bottle is discarded, the culture solution is added into the culture bottle, continues culture 3-10 days
Afterwards to get to mescenchymal stem cell.Every group of mescenchymal stem cell yield is counted, as a result sees Fig. 3, wherein, G is what G groups obtained
The quantity of mescenchymal stem cell；H is the quantity of mescenchymal stem cell that H groups obtain；I is the mescenchymal stem cell that I groups obtain
Quantity.From the figure 3, it may be seen that the quantity for the mescenchymal stem cell that G groups obtain is 5 × 105A/cm；H groups obtain mescenchymal stem cell
Quantity is 1 × 105A/cm；The quantity that I groups obtain mescenchymal stem cell is 6 × 104A/cm.H groups are compared with I group data, are shown
Compared with prior art, more mescenchymal stem cells can be obtained using the present embodiment method, is organized after recovery can be effectively increased
Activity, improve the yield of mescenchymal stem cell.
Basic principle, main feature and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent defines.
Those skilled in the art are supplied to the purpose described to the description of the various embodiments of the present invention above.It is not
It is intended to exhaustive or is not intended to and limits the invention to single disclosed embodiment.As described above, the present invention's is various
It substitutes and variation will be apparent for above-mentioned technology one of ordinary skill in the art.Therefore, although specifically begging for
Some alternative embodiments are discussed, but other embodiment will be apparent or those skilled in the art are opposite
Easily obtain.The present invention is directed to all replacements of the present invention, modification and the variation that include having discussed herein and fall
Other embodiment in the spirit and scope of above-mentioned application.
Although depicting the present invention by embodiment, it will be appreciated by the skilled addressee that there are many deform by the present invention
With variation without departing from the spirit of the present invention, it is desirable to which appended claim includes these deformations and changes without departing from the present invention
Spirit.
Claims (15)
- A kind of 1. umbilical cord tissue cryopreservation resuscitation method, which is characterized in that including：Step S1 obtains umbilical cord tissue, cuts the umbilical cord tissue to obtain the first tissue item；Step S2, the first tissue item is immersed in vetrifying solution, then directly freezes the first tissue item to obtain Minor microstructure item；Step S3 recovers the minor microstructure item to obtain third strip of tissue；The vetrifying solution includes the first vetrifying solution, the second vetrifying solution and third vetrifying solution, and the step S2 includes will The first tissue item is immersed in first vetrifying solution, second vetrifying solution and the third vetrifying solution successively In, the first tissue item is then moved into freezen protective in liquid nitrogen, obtains the minor microstructure item.
- 2. the cryopreservation resuscitation method of umbilical cord tissue according to claim 1, which is characterized in that the umbilical cord tissue includes sheep Film layer, the step S1 include the rete layer for tearing off the umbilical cord tissue.
- 3. the cryopreservation resuscitation method of umbilical cord tissue according to claim 1, which is characterized in that the umbilical cord tissue includes navel Artery and umbilical vein, the step S1 include the arteria umbilicalis for tearing off the umbilical cord tissue, retain the institute of the umbilical cord tissue State umbilical vein.
- 4. the cryopreservation resuscitation method of umbilical cord tissue according to claim 1, which is characterized in that the first tissue item is in institute The soaking time stated in the first vetrifying solution, second vetrifying solution and the third vetrifying solution is 2-7min, described The temperature of first vetrifying solution, second vetrifying solution and the third vetrifying solution is 2-6 DEG C.
- 5. the cryopreservation resuscitation method of umbilical cord tissue according to claim 4, which is characterized in that the third vetrifying solution packet Impermeability protective agent, permeability protective agent, thickener, KSR serum and DMEM culture mediums are included, the impermeability protective agent A concentration of 0.4-1.0mol/L, the protectant percent by volume of permeability be 10-30%, the volume basis of the thickener Than being 10-20% for the percent by volume of 0.5-5%, the KSR serum.
- 6. the cryopreservation resuscitation method of umbilical cord tissue according to claim 5, which is characterized in that the permeability protective agent packet Dimethyl sulfoxide (DMSO) or ethylene glycol are included, the impermeability protective agent includes any one in sucrose, glucose or trehalose Or it is a variety of, it is fine that the thickener includes chondroitin sulfate, dextran, Sodium Hyaluronate, carboxylic propyl methocel, carboxymethyl Tie up any one or more in element, polyvinylpyrrolidone, POLYPROPYLENE GLYCOL or polyvinyl alcohol.
- 7. the cryopreservation resuscitation method of umbilical cord tissue according to claim 1 or 5, which is characterized in that second vitrifying The component of liquid presses volume percentage, including：It is third vetrifying solution 40%-60%, KSR serum substitute 10-20%, remaining It measures as DMEM culture mediums.
- 8. the cryopreservation resuscitation method of umbilical cord tissue according to claim 1 or 5, which is characterized in that first vitrifying The component of liquid presses volume percentage, including：It is third vetrifying solution 20%-30%, KSR serum substitute 10-20%, remaining It measures as DMEM culture mediums.
- 9. the cryopreservation resuscitation method of umbilical cord tissue according to claim 1, which is characterized in that the step S3 is included with multiple Liquid of reviving handles the minor microstructure item, and the resuscitation fluid includes the first resuscitation fluid, the second resuscitation fluid and third resuscitation fluid, the place Reason process is answered including the minor microstructure item is immersed in first resuscitation fluid, second resuscitation fluid and the third successively In Soviet Union's liquid.
- 10. the cryopreservation resuscitation method of umbilical cord tissue according to claim 9, which is characterized in that the minor microstructure item exists The immersion treatment time in first resuscitation fluid, second resuscitation fluid and the third resuscitation fluid is 2-15min, described The temperature of first resuscitation fluid, second resuscitation fluid and the third resuscitation fluid is 20-38 DEG C.
- 11. the cryopreservation resuscitation method of the umbilical cord tissue according to claim 5 or 9, which is characterized in that first resuscitation fluid Including glucide, KSR serum and DMEM culture mediums, a concentration of 0.4-1.0mol/L of the glucide, the KSR serum The percent by volume of substitute be 10-20%, the glucide include sucrose, trehalose or glucose in any one or It is a variety of；The component of second resuscitation fluid presses volume percentage, including：First resuscitation fluid 40%-60%, KSR serum replaces It is DMEM culture mediums for object 10-20%, surplus；The component of the third resuscitation fluid presses volume percentage, including：KSR serum substitute 10-20%, surplus are trained for DMEM Support base.
- 12. the cryopreservation resuscitation method of umbilical cord tissue according to claim 1, which is characterized in that the step S3 includes using Third strip of tissue described in culture solution culture.
- 13. the cryopreservation resuscitation method of the umbilical cord tissue according to claim 5 or 12, which is characterized in that the culture solution packet Include vitamin, tert-butyl hydroquinone, Caspase inhibitors, N-acetyl-L-cysteine, DMEM culture mediums With KSR serum substitutes.
- 14. a kind of separation method of mescenchymal stem cell, which is characterized in that include the following steps：It obtains and uses claim 1- The third strip of tissue that 13 any one of them umbilical cord tissue cryopreservation resuscitation methods obtain, trains the third strip of tissue successively It supports with digestion process to form cell suspension and fragment of tissue, remove the fragment of tissue and cultivate the cell suspension to be formed The mescenchymal stem cell.
- 15. the separation method of mescenchymal stem cell according to claim 14, which is characterized in that the tissue pieces group It knits digestive juice and carries out the digestion process, the tissue digestion liquid is the mixture of clostridiopetidase A, dispase and hyaluronidase.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108990964A (en) * | 2018-08-09 | 2018-12-14 | 华东理工大学 | Cells frozen storing liquid |
| CN109042628A (en) * | 2018-09-21 | 2018-12-21 | 洛阳未羊生物科技有限公司 | A kind of cryopreservation methods of amnion tissue |
| CN109392891A (en) * | 2018-10-26 | 2019-03-01 | 银丰生物工程集团有限公司 | A kind of methods and applications freezing human umbilical tissue according to layer of structure system |
| CN109497039A (en) * | 2018-10-29 | 2019-03-22 | 上海慧存医疗科技有限公司 | The cryopreservation resuscitation method of umbilical cord tissue and the preparation method of mescenchymal stem cell |
| CN110250165A (en) * | 2019-07-24 | 2019-09-20 | 安徽科门生物科技有限公司 | A kind of umbilical cord mesenchymal stem cells frozen stock solution and cryopreservation methods |
| CN110551684A (en) * | 2019-09-16 | 2019-12-10 | 福建省海西细胞生物工程有限公司 | preparation method of human umbilical cord mesenchymal stem cells |
| CN110800733A (en) * | 2019-11-21 | 2020-02-18 | 武汉光谷中源协和细胞基因科技有限公司 | Cryopreservation solution and kit for umbilical cord mesenchymal stem cells |
| CN111280166A (en) * | 2020-04-08 | 2020-06-16 | 广州裕康生物科技有限公司 | Vitrification refrigerating fluid and freezing method of blastocyst |
| WO2020165152A1 (en) * | 2019-02-13 | 2020-08-20 | Tigenix, S.A.U. | Cryopreservation of stem cells |
| CN113303323A (en) * | 2020-02-27 | 2021-08-27 | 东莞市恩联干细胞生物科技研究院 | Non-freezing preservation method for umbilical cord tissue |
| CN116889228A (en) * | 2023-07-12 | 2023-10-17 | 重庆市铂而斐细胞生物技术有限公司 | Cryopreservation method of umbilical cord mesenchymal stem cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104145943A (en) * | 2014-07-21 | 2014-11-19 | 重庆市红汇脐血干细胞中心有限公司 | Cryopreservation protection liquid for Wharton jelly tissues of human umbilical cord and preparation and application of cryopreservation protection liquid |
| US20160066566A1 (en) * | 2014-09-10 | 2016-03-10 | Healthbanks Biotech Co., Ltd. | Cryopreservation of umbilical cord tissue strips for cord tissue-derived stem cells |
-
2017
- 2017-12-25 CN CN201711424190.XA patent/CN108130308B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104145943A (en) * | 2014-07-21 | 2014-11-19 | 重庆市红汇脐血干细胞中心有限公司 | Cryopreservation protection liquid for Wharton jelly tissues of human umbilical cord and preparation and application of cryopreservation protection liquid |
| US20160066566A1 (en) * | 2014-09-10 | 2016-03-10 | Healthbanks Biotech Co., Ltd. | Cryopreservation of umbilical cord tissue strips for cord tissue-derived stem cells |
Non-Patent Citations (2)
| Title |
|---|
| LILIAN DA-CROCE ET AL.: "Comparison of vitrification and slow cooling for umbilical tissues", 《CELL TISSUE BANK》 * |
| SHARATH BELAME SHIVAKUMAR ET AL.: "Cryopreservation of Human Wharton’s Jelly-derived Mesenchymal Stem Cells Following Controlled Rate Freezing Protocol Using Different Cryoprotectants; A Comparative Study", 《INTERNATIONAL JOURNAL OF STEM CELLS》 * |
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|---|---|---|---|---|
| CN108990964A (en) * | 2018-08-09 | 2018-12-14 | 华东理工大学 | Cells frozen storing liquid |
| CN109042628A (en) * | 2018-09-21 | 2018-12-21 | 洛阳未羊生物科技有限公司 | A kind of cryopreservation methods of amnion tissue |
| CN109392891A (en) * | 2018-10-26 | 2019-03-01 | 银丰生物工程集团有限公司 | A kind of methods and applications freezing human umbilical tissue according to layer of structure system |
| CN109497039A (en) * | 2018-10-29 | 2019-03-22 | 上海慧存医疗科技有限公司 | The cryopreservation resuscitation method of umbilical cord tissue and the preparation method of mescenchymal stem cell |
| CN113423268B (en) * | 2019-02-13 | 2024-04-02 | 武田药品工业株式会社 | Cryopreservation of stem cells |
| WO2020165152A1 (en) * | 2019-02-13 | 2020-08-20 | Tigenix, S.A.U. | Cryopreservation of stem cells |
| CN113423268A (en) * | 2019-02-13 | 2021-09-21 | 泰根尼克斯独资有限公司 | Cryopreservation of stem cells |
| CN110250165A (en) * | 2019-07-24 | 2019-09-20 | 安徽科门生物科技有限公司 | A kind of umbilical cord mesenchymal stem cells frozen stock solution and cryopreservation methods |
| CN110551684B (en) * | 2019-09-16 | 2021-09-03 | 福建省海西细胞生物工程有限公司 | Preparation method of human umbilical cord mesenchymal stem cells |
| CN110551684A (en) * | 2019-09-16 | 2019-12-10 | 福建省海西细胞生物工程有限公司 | preparation method of human umbilical cord mesenchymal stem cells |
| CN110800733A (en) * | 2019-11-21 | 2020-02-18 | 武汉光谷中源协和细胞基因科技有限公司 | Cryopreservation solution and kit for umbilical cord mesenchymal stem cells |
| CN113303323A (en) * | 2020-02-27 | 2021-08-27 | 东莞市恩联干细胞生物科技研究院 | Non-freezing preservation method for umbilical cord tissue |
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| CN116889228A (en) * | 2023-07-12 | 2023-10-17 | 重庆市铂而斐细胞生物技术有限公司 | Cryopreservation method of umbilical cord mesenchymal stem cells |
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