CN102660502A - Methods for freezing and thawing whole cell of umbilical cord and separating and augmenting stem cell - Google Patents

Methods for freezing and thawing whole cell of umbilical cord and separating and augmenting stem cell Download PDF

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CN102660502A
CN102660502A CN2012101599196A CN201210159919A CN102660502A CN 102660502 A CN102660502 A CN 102660502A CN 2012101599196 A CN2012101599196 A CN 2012101599196A CN 201210159919 A CN201210159919 A CN 201210159919A CN 102660502 A CN102660502 A CN 102660502A
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cell
umbilical cord
frozen
tissue
stem cell
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CN102660502B (en
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林卓衡
陈俊峯
朱业峰
周丹
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BOYA STEM CELL TECHNOLOGY Co Ltd
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BOYA STEM CELL TECHNOLOGY Co Ltd
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Abstract

The invention relates to methods for separating, freezing and thawing a whole cell of an umbilical cord and separating and augmenting a thawed stem cell. The method for separating and freezing the whole cell of the umbilical cord comprises the following steps of: sterilizing and cleaning an umbilical cord tissue; shearing the tissue into a block shape and carrying out digestion treatment for 1.5 hours; preparing a umbilical cord tissue frozen solution for later use; and adding the whole cell and frozen solution which are obtained by sterilizing treatment into a freezing tube, refrigerating the freezing tube for 0.5 hour at low temperature of 4DEG C, freezing the freezing tube for one day under the temperature condition of -80DEG C and then freezing the freezing tube in liquid nitrogen for later use. The method for thawing the whole cell of the umbilical cord comprises the following steps of: when the whole cell of the umbilical cord is required, extracting the whole cell of the umbilical cord from the liquid nitrogen, thawing the whole cell of the umbilical cord in a constant-temperature water bath; cleaning the whole cell of the umbilical cord by using a mesenchymal stem cell culture medium and a drop method; and augmenting a mesenchymal stem cell by using the thawed whole cell of the umbilical cord through cell culture and cell passage. According to the methods disclosed by the invention, the frozen umbilical cord tissue can be effectively protected and is convenient for thawing; and the method is especially suitable for separating and augmenting the mesenchymal stem cell after the frozen umbilical cord tissue is thawed.

Description

Frozen and the full cell of recovery umbilical cord also separate and the method for expanding stem cells
Technical field
The present invention relates to method that the full cell of umbilical cord is handled; Be specifically related to the full cell cryopreservation of umbilical cord, recovery are handled; And by the full cellular segregation of umbilical cord after the recovery and the method for expanding stem cells; Be particularly related to the full cell of umbilical cord is carried out frozen, recovery, and then therefrom separate and the method for amplification of mesenchymal stem cells.
Background technology
(mesenchymal stem cells MSC) derives from growth early stage mesoderm and ectoderm to mescenchymal stem cell, has characteristics such as multidirectional differentiation potential, immunomodulatory and self-replacation, receives people's attention day by day.Mescenchymal stem cell is in vivo or under the external specific inductive condition; Can be divided into multiple histocytes such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium; Still has multidirectional differentiation potential after continuous passage cultivation and the freezing preservation; Can be used as the ideal seed cell and be used for injuries of tissues and organs reparation old and feeble and that pathology causes, especially very big clinical value is arranged treating aging and injuries of tissues and organs reparation.
MSC contains abundant in marrow, but aging along with the age, the stem cell number in the marrow also can significantly reduce, the also decline significantly of proliferation and differentiation ability.In addition; Marrow MSC transplants to allosome possibly cause immunoreation; And extract the stem cell process to the damaging of patient and the other problems that when gathering, runs into; All directly influenced the clinical application of marrow MSC, made that seeking marrow becomes an important problem in other alternative mescenchymal stem cells sources in addition.
Recent research shows, also contains mescenchymal stem cell in the umbilical cord tissue and can successfully separate.This tissue-derived mescenchymal stem cell has not only kept the biological characteristics of mescenchymal stem cell, and the stem cell of separating is more original, and stronger proliferation and differentiation ability is arranged.The functionally active of its immunocyte is low, has lowered the risk that triggers immunoreation and cause graft versus host disease greatly.Occult virus and infection by microorganisms and propagation probability are lower.Gatherer process is simple, and puerpera and newborn infant are not had any harm and damage.Above reason is enough to make umbilical cord mesenchymal stem cells to become the desirable surrogate of mesenchymal stem cells MSCs.
But umbilical cord tissue for example method and the technology of the full cellular segregation stem cell of umbilical cord also is not a fully matured, and the processing of the full cell of each part umbilical cord with separate after cell cultures all need regular hour and personnel's consumption.Therefore the way of cultivating and increasing of recovering with the full cell cryopreservation of umbilical cord and when needs are arranged relatively more meets cost benefit.Therefore, this area needs the method for simple and the full cellular segregation of umbilical cord efficiently, frozen, recovery and amplification, to satisfy the demand in medicine, scientific research, field such as clinical.
Summary of the invention
The objective of the invention is to solve that prior art is frozen, the defective of the full cell method of recovery umbilical cord; The method of the full cell cryopreservation of a kind of simple and effective umbilical cord is provided; And the method for the full cell recovery of freezing and storing umbilical of the use that matches, and the method for recovery back mescenchymal stem cell amplification.The present invention finds the full cell of umbilical cord is used frozen, the recovery of specific operation step and separates and amplification; Can be simply, effectively from the umbilical cord tissue full cellular segregation of umbilical cord mescenchymal stem cell of former generation for example, and can in frozen process, protect effectively former generation mescenchymal stem cell.The present invention is based on this discovery and be accomplished.
Therefore, first aspect present invention provides the method for handling the full cell of umbilical cord, and this method comprises followingly separates and frozen step the umbilical cord full cell of flesh tissue that exsomatizes:
(1) sterilization and cleaning: with thimerosal (for example alcohol) the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off, tiling is cleaned umbilical cord tissue through damping fluid (for example PBS damping fluid), to reduce red corpuscle on the umbilical cord tissue;
(2) digestion process: the umbilical cord tissue that step (1) is obtained is cut into tissue block; Tissue block is put into digestive ferment solution (for example it comprises type i collagen enzyme, DMEM-F12 without limitation); Digestion process 0.5-3 hour (for example 1.5 hours); Filter and remove tissue block, add the mescenchymal stem cell substratum to stop digestion, the cell that then digestion is obtained carries out cell and cleans;
(3) preparation umbilical cord full cells frozen storing liquid: comprise rHSA and DMSO (DMSO 99.8MIN.) in the full cells frozen storing liquid of said umbilical cord, the cold liquid storage for preparing is placed on deepfreeze under the temperature condition of 1 ℃ to 7 ℃ (for example 4 ℃);
(4) cold the depositing of the full cell of umbilical cord: at 0-15 ℃ (for example 1 ℃ to 7 ℃; For example 4 ℃) low temperature environment under; With re-suspended cell, then re-suspended cell is added in the frozen container in the cell after the cleaning that the full cells frozen storing liquid adding step (2) that step (3) is obtained obtains, frozen container is put into the programmed cooling device; Earlier under the temperature condition of 1 ℃ to 7 ℃ (for example 4 ℃) deepfreeze 0.2-2 hour (for example 0.5 hour); Under the temperature condition of-10 ℃ to-150 ℃ (for example-80 ℃) freezing 0.25-3 days (for example 1 day), frozen container is freezing in liquid nitrogen, subsequent use then again.
According to the method for first aspect present invention, this method also comprises the following step that the stripped full cell of flesh tissue of frozen umbilical cord is recovered:
(5) the full cell recovery of freezing and storing umbilical: the full cell of step (4) refrigerated umbilical cord is taken out from liquid nitrogen, and thawing to 20%-70% (for example 50%) frozen storing liquid begins to melt, and utilizes the mescenchymal stem cell substratum to clean cell, so that the full cell recovery of frozen umbilical cord.
According to the method for first aspect present invention, this method comprises that also the following full cell of umbilical cord to recovery carries out the step that mescenchymal stem cell separates and increases:
(6) cell cultures: in the cell that step (5) obtains, add the mescenchymal stem cell substratum of an amount of (for example 1-6 times of cell suspension volume amount), put into culture vessel, culture vessel is put in the incubator cultivated again; Be cultured to 2-7 days and culture vessel taken out from incubator when (for example 3-6 days, for example the 4th day, for example the 5th day); Add an amount of (for example 0.2-2 times of enchylema volume) mescenchymal stem cell substratum; Continue to cultivate, when 8-11 days (for example the 9th day), culture vessel is taken out from incubator, carry out changing full the first time liquid; Continue to cultivate, (for example 2 days) were once changed liquid entirely in every backward 1-3 days;
(7) passage: after the attached cell fusion rate in the culture vessel reaches 40%-70% (for example 60%); Utilize digestive ferment (for example TrypLe Express) that attached cell is broken away from container bottom, centrifugal, take supernatant away; Add mesenchymal stem cells substratum suspension cell again; Be inoculated in that culture vessel goes down to posterity and carry out amplification cultivation, liquid was changed once in (for example per 2 days) in after this every 1-3 days, after fusion rate reaches 70-90% (for example 80%); Promptly get umbilical cord mesenchymal stem cells, go down to posterity in case of necessity;
And optional following one or more steps:
(8) to being directed against step (7) gained umbilical cord mesenchymal stem cells, detect at least one item of following project: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type;
(9) umbilical cord mesenchymal stem cells after step (7) gained is gone down to posterity is freezing in liquid nitrogen, subsequent use.
According to the method for first aspect present invention, this method comprises:
(A) below the stripped full cell of flesh tissue of umbilical cord is separated and frozen step:
(1) sterilization and cleaning: with thimerosal (for example alcohol) the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off, tiling is cleaned umbilical cord tissue through damping fluid (for example PBS damping fluid), to reduce red corpuscle on the umbilical cord tissue;
(2) digestion process: the umbilical cord tissue that step (1) is obtained is cut into tissue block; Tissue block is put into digestive ferment solution (for example it comprises type i collagen enzyme, DMEM-F12 without limitation); Digestion process 0.5-3 hour (for example 1.5 hours); Filter and remove tissue block, add the mescenchymal stem cell substratum to stop digestion, the cell that then digestion is obtained carries out cell and cleans;
(3) preparation umbilical cord full cells frozen storing liquid: comprise rHSA and DMSO (DMSO 99.8MIN.) in the full cells frozen storing liquid of said umbilical cord, the cold liquid storage for preparing is placed on deepfreeze under the temperature condition of 1 ℃ to 7 ℃ (for example 4 ℃);
(4) cold the depositing of the full cell of umbilical cord: at 0-15 ℃ (for example 1 ℃ to 7 ℃; For example 4 ℃) low temperature environment under; With re-suspended cell, then re-suspended cell is added in the frozen container in the cell after the cleaning that the full cells frozen storing liquid adding step (2) that step (3) is obtained obtains, frozen container is put into the programmed cooling device; Earlier under the temperature condition of 1 ℃ to 7 ℃ (for example 4 ℃) deepfreeze 0.2-2 hour (for example 0.5 hour); Under the temperature condition of-10 ℃ to-150 ℃ (for example-80 ℃) freezing 0.25-3 days (for example 1 day), frozen container is freezing in liquid nitrogen, subsequent use then again;
The step of (B) the stripped full cell of flesh tissue of frozen umbilical cord being recovered:
(5) the full cell recovery of freezing and storing umbilical: the full cell of step (4) refrigerated umbilical cord is taken out from liquid nitrogen, and thawing to 20%-70% (for example 50%) frozen storing liquid begins to melt, and utilizes the mescenchymal stem cell substratum to clean cell, so that the full cell recovery of frozen umbilical cord;
(C) the full cell of umbilical cord to recovery below carries out the step that mescenchymal stem cell separates and increases:
(6) cell cultures: in the cell that step (5) obtains, add the mescenchymal stem cell substratum of an amount of (for example 1-6 times of cell suspension volume amount), put into culture vessel, culture vessel is put in the incubator cultivated again; Be cultured to 2-7 days and culture vessel taken out from incubator when (for example 3-6 days, for example the 4th day, for example the 5th day); Add an amount of (for example 0.2-2 times of enchylema volume) mescenchymal stem cell substratum; Continue to cultivate, when 8-11 days (for example the 9th day), culture vessel is taken out from incubator, carry out changing full the first time liquid; Continue to cultivate, (for example 2 days) were once changed liquid entirely in every backward 1-3 days;
(7) passage: after the attached cell fusion rate in the culture vessel reaches 40%-70% (for example 60%); Utilize digestive ferment (for example TrypLe Express) that attached cell is broken away from container bottom, centrifugal, take supernatant away; Add mesenchymal stem cells substratum suspension cell again; Be inoculated in that culture vessel goes down to posterity and carry out amplification cultivation, liquid was changed once in (for example per 2 days) in after this every 1-3 days, after fusion rate reaches 70-90% (for example 80%); Promptly get umbilical cord mesenchymal stem cells, go down to posterity in case of necessity;
And optional
(D) following one or more step:
(8) to being directed against step (7) gained umbilical cord mesenchymal stem cells, detect at least one item of following project: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type;
(9) umbilical cord mesenchymal stem cells after step (7) gained is gone down to posterity is freezing in liquid nitrogen, subsequent use.
According to the method for first aspect present invention, wherein said umbilical cord is the fresh in vitro tissue of umbilical cord.
According to the method for first aspect present invention, wherein the said umbilical cord tissue of step (1) is in Biohazard Safety Equipment, to handle.
According to the method for first aspect present invention, wherein the step of the said tiling of step (1) is that umbilical cord tissue is tiled in the culture dish, and said culture dish diameter is the culture dish of 5-20cm, and preferred petridish diameter is the culture dish of 10cm.
According to the method for first aspect present invention, the concentration of wherein said alcohol is 25%-95%, preferred 75%.
According to the method for first aspect present invention, sodium salt that wherein said PBS damping fluid is a phosphoric acid and/or sylvite preparation, its pH is 5.0-8.0, and preferred pH is 5.5-76, and preferred pH is 6.0-7.0.In one embodiment, the concentration of phosphate radical is 0.01-0.5M in the said PBS damping fluid, preferred 0.02-0.1M.In hereinafter test of the present invention, used PBS damping fluid is a sodium phosphate salt, and wherein the concentration of phosphate radical is 0.025M, and pH is 6.5.Need to prove that the inventor finds that PBS buffer concentration and pH value in above-mentioned scope are little for the influential effect of the inventive method.
According to the method for first aspect present invention, wherein said digestive ferment solution is that the type i collagen enzyme is added DMEM-F12, obtains through the strainer filtration; Its digestive ferment is 0.05g-0.5g, and preferred digestive ferment is 0.08g-0.2g, and preferred digestive ferment is 0.1g; Its DMEM-F12 is 50-500ml, and preferred DMEM-F12 is 80-200ml, and preferred DMEM-F12 is 100ml; Its strainer is a 5-50 μ m strainer, preferred 20 μ m strainers.In one embodiment, said digestive ferment solution is that the type i collagen enzyme with 0.1g joins among the DMEM-F12 of 100ml, and mixing filters that (for example with the filtration of 20um strainer) obtain.
Method according to first aspect present invention; Wherein step (2) is that umbilical cord tissue is transferred in another cell cultures plate; Carry out umbilical cord tissue then and be cut into tissue block, said culture dish diameter is the culture dish of 5-20cm, and preferred petridish diameter is the culture dish of 10cm.
According to the method for first aspect present invention, wherein in the step (2), the size of tissue block is about the 0.2-2.5 cubic centimetre, preferably is about the 0.5-1.5 cubic centimetre, and preferred about 1 cubic centimetre cube is block.
According to the method for first aspect present invention, wherein in the step (2), the tissue block size is at the 0.5-1.5 cubic centimetre, and preferred 0.5-1.0 cubic centimetre is very preferred in the time of particularly big or small about 1 cubic centimetre.Although the intended tissue fragment helps the realization of the inventive method for a short time; Yet the inventor finds in test under 0.2 cubic centimetre, 0.5 cubic centimetre, 1 cubic centimetre three kinds of states; They are to the digestion process effect basically identical of digestive ferment; And volume has remarkable disadvantageous effect greater than 1.5 cubic centimetres of later digestion effects to digestive ferment, and this disadvantageous effect can weaken through prolonging digestion time to a certain extent.
According to the method for first aspect present invention, wherein in the step (2), the time of digestion process is 0.5-3 hour, preferred 1-2.5 hour, and preferred 1.5-2 hour.The inventor finds 1-2.5 hour digestion process in the time, is best to the digestion process effect of tissue block, both can guarantee that tissue block obtains sufficient digestion process, also can avoid cell to be destroyed.
According to the method for first aspect present invention, wherein in the step (2), digestion process is to carry out near the TR body temperature, preferred 34-40 ℃, and preferred 36-38 ℃, preferred 37 ℃.
According to the method for first aspect present invention, wherein in the step (2), digestion process is carried out in the constant temperature shaking table.
According to the method for first aspect present invention, wherein in the step (2), filter the removing tissue block and carry out through filter screen, said filter screen is a 50-150 μ m filter screen, preferred 100 μ m filter screens.
According to the method for first aspect present invention, wherein in the step (2), the mescenchymal stem cell substratum that stops digestion is the ratio adding according to 2:1 ~ 1:2, the ratio of preferred 1:1, and said ratio is a volume ratio.
According to the method for first aspect present invention, wherein in the step (2), the concrete steps that cell cleans are centrifugal 5-15 minute, remove supernatant, add PBS damping fluid re-suspended cell, centrifugal again 5-15 minute, remove supernatant.Centrifugal rotational speed is 800-2000rpm, preferred 1250rpm, and centrifugation time is preferred 10 minutes.
According to the method for first aspect present invention, comprise FBS, L-Glutamine (L-L-glutamic acid), Gentamicin (qingfengmeisu qiong) and DMEM-F12 in the wherein said mescenchymal stem cell substratum.In one embodiment, the FBS that contains 10-20% in the said mescenchymal stem cell substratum.In one embodiment, contain 15% the FBS of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, the L-Glutamine that contains 0.5-2% in the said mescenchymal stem cell substratum.In one embodiment, contain 1% the L-Glutamine of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, contain 0.05% the Gentamicin of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, contain 0.05% the Gentamicin of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, the DMEM-F12 that contains 80-90% in the said mescenchymal stem cell substratum.In one embodiment, contain 84% the DMEM-F12 of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, contain the Gentamicin of the L-Glutamine of the FBS of 15 weight parts of having an appointment, about 1 weight part, about 0.05 weight part and the DMEM-F12 of about 84 weight parts in the said mescenchymal stem cell substratum.The inventor finds; The mescenchymal stem cell substratum of DMEM-F12 that contains Gentamicin and about 84 weight parts of the L-Glutamine of the FBS of 15 weight parts of having an appointment, about 1 weight part, about 0.05 weight part is preferred especially, than the arbitrary component content in making this substratum change prescription 10% or more adherent at the increase umbilical cord tissue, shorten and have significant advantage aspect the effects such as time that attached cell climbs out of from tissue.
According to the method for first aspect present invention, comprise rHSA and DMSO in the wherein said umbilical cord tissue frozen storing liquid.In one embodiment, the rHSA that contains the 55-95 weight part in the said umbilical cord tissue frozen storing liquid.In one embodiment, the rHSA that contains the 70-90 weight part in the said umbilical cord tissue frozen storing liquid.In one embodiment, the rHSA that contains 80 weight parts in the said umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains the 4-20 weight part in the said umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains the 7-15 weight part in the said umbilical cord tissue frozen storing liquid.In one embodiment, the DMSO that contains 10 weight parts in the said umbilical cord tissue frozen storing liquid.In one embodiment, contain the rHSA of 80 weight parts of having an appointment, the DMSO of about 10 weight parts in the said umbilical cord tissue frozen storing liquid.In the above-mentioned embodiment, the summation of each component concentration is 100% in the umbilical cord tissue frozen storing liquid.The inventor finds; The umbilical cord tissue frozen storing liquid (promptly wherein the weight ratio of rHSA and DMSO is 80:10) that contains the DMSO of the rHSA of 80 weight parts of having an appointment, about 10 weight parts is preferred especially, than the prescription that changes 10% or more in the arbitrary component content that makes this umbilical cord tissue frozen storing liquid protecting umbilical cord tissue not receive to have significant advantage aspect the effect such as refrigerating process destruction.In the present invention, owing to also possibly contain those skilled in the art general other dosing solvent or solute in the frozen storing liquid, therefore above-mentioned is a kind of relative quantity with rHSA of " weight part " expression and the amount of DMSO, and it can be milligram, gram, kilogram etc.
According to the method for first aspect present invention, wherein the said full cells frozen storing liquid that step (3) is obtained of step (4) adds cell after the cleaning that step (2) obtains with re-suspended cell, extracts sample behind the re-suspended cell and carries out cell counting.The add-on of full cells frozen storing liquid is 1-4 a times of enchylema volume, for example 2 times.
According to the method for first aspect present invention, wherein the said low temperature environment of step (4) is 0-15 ℃, preferred 1 ℃ to 7 ℃, and preferred 4 ℃.
According to the method for first aspect present invention, wherein the said frozen container of step (4) is frozen pipe.
According to the method for first aspect present invention, the volume of the re-suspended cell frozen storing liquid that wherein adds in said each frozen container of step (4) (frozen pipe) is 0.5-3ml, preferred 1ml.
According to the method for first aspect present invention, wherein the density of the described cell of step (4) in frozen container is the upper limit to guarantee that arbitrary cell all has enough DMSO that protection is provided, and said density is meant the cell individual number in the unit space, preferred 1 * 10 3-1 * 10 8Every pipe, preferred 1 * 10 6Every pipe.
According to the method for first aspect present invention, wherein the described programmed cooling device of step (4) is the programmed cooling box.
According to the method for first aspect present invention, wherein described deepfreeze of step (4) and freezing are put into the refrigerator realization with programmed cooling device (for example programmed cooling box).
According to the method for first aspect present invention, wherein the described sample of step (4) refrigeration flow process is under 1 ℃ to 7 ℃ temperature condition deepfreeze 0.2-2 hour.In one embodiment, said deepfreeze is under 2 ℃ to 6 ℃ temperature condition.In one embodiment, said deepfreeze is under 3 ℃ to 5 ℃ temperature condition.In one embodiment, said deepfreeze is under 5 ℃ temperature condition.In one embodiment, the said deepfreeze time is 0.3-1.5 hour.In one embodiment, the said deepfreeze time is 0.4-1 hour.In one embodiment, the said deepfreeze time is 0.5 hour.The inventor finds that deepfreeze 0.5 hour was preferred especially under 4 ℃ temperature condition, can guarantee that the full cell of DMSO and umbilical cord fully merges.Above-mentioned other refrigerating temperature conditions and cold preservation time also can make the full cytogamy of DMSO and umbilical cord, and its syncretizing effect can satisfy primary demand of the present invention, but 0.5 hour syncretizing effect fullest of deepfreeze under 4 ℃ the temperature condition has clear superiority.
According to the method for first aspect present invention, wherein the freezing flow process of the described sample of step (4) is under-10 ℃ to-150 ℃ temperature condition freezing 0.25-3 days.In one embodiment, said freezing be under-30 ℃ to-120 ℃ temperature condition.In one embodiment, said freezing be under-50 ℃ to-100 ℃ temperature condition.In one embodiment, said freezing be under-80 ℃ temperature condition.In one embodiment, said freezing time is 0.4-2 days.In one embodiment, said freezing time is 0.8-1.5 days.In one embodiment, said freezing time is 1 day.The inventor finds; Freezing 1 day is preferred especially under-80 ℃ temperature condition, than the freezing flow process that changes 10% or more in the arbitrary temperature that makes this freezing flow process, time protecting umbilical cord tissue not receive to have significant advantage aspect the effect such as refrigerating process destruction.
According to the method for first aspect present invention, wherein said the thawing of step (5) is in water bath with thermostatic control, to carry out.
According to the method for first aspect present invention, comprise FBS, L-Glutamine, Gentamicin and DMEM-F12 in the wherein said mescenchymal stem cell substratum.In one embodiment, the FBS that contains 10-20% in the said mescenchymal stem cell substratum.In one embodiment, contain 15% the FBS of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, the L-Glutamine that contains 0.5-2% in the said mescenchymal stem cell substratum.In one embodiment, contain 1% the L-Glutamine (L-glutaminate) of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, the Gentamicin (qingfengmeisu qiong) that contains 0.02-0.1% in the said mescenchymal stem cell substratum.In one embodiment, contain 0.05% the Gentamicin of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, the DMEM-F12 that contains 80-90% in the said mescenchymal stem cell substratum.In one embodiment, contain 84% the DMEM-F12 of having an appointment in the said mescenchymal stem cell substratum.In one embodiment, contain the Gentamicin of the L-Glutamine of the FBS of 15 weight parts of having an appointment, about 1 weight part, about 0.05 weight part and the DMEM-F12 of about 84 weight parts in the said mescenchymal stem cell substratum.The inventor finds; The mescenchymal stem cell substratum of DMEM-F12 that contains Gentamicin and about 84 weight parts of the L-Glutamine of the FBS of 15 weight parts of having an appointment, about 1 weight part, about 0.05 weight part is preferred especially, than the arbitrary component content in making this substratum change prescription 10% or more adherent at the increase umbilical cord tissue, shorten and have significant advantage aspect the effects such as time that attached cell climbs out of from tissue.
According to the method for first aspect present invention, wherein the said mescenchymal stem cell substratum of step (5) is to clean cell through topical application.Topical application can clean out in-house DMSO effectively, thereby avoids the loss of cell survival rate in the recovery process.
According to the method for first aspect present invention, wherein the volume ratio of said cell suspension of step (5) and mescenchymal stem cell substratum is 1:2-1:5, and preferred volume ratio is 1:3.
According to the method for first aspect present invention, wherein the said culture vessel of step (6) is the T25 Tissue Culture Flask.
According to the method for first aspect present invention, CO in the said incubator of step (6) wherein 2Concentration is 3-7%, and preferred concentration is 5%, and the incubator temperature is controlled in the body temperature environs, preferred 34-40 ℃, and preferred 36-38 ℃, preferred 37 ℃.
According to the method for first aspect present invention, in step (8), it is to utilize the trypan blue staining to count the number of frozen front and back viable cell that said cytoactive detects.
According to the method for first aspect present invention, in step (8), said cell contamination detects and utilizes small amounts of cells to cultivate, and detects cell and whether receives the pollution of fungi and bacterium.In one embodiment; Whether it is to utilize the etiology method that said cell contamination detects, detect cell and receive and be selected from following one or multinomial infection: hepatitis B two double, third liver, virus of AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST.
According to the method for first aspect present invention, in step (8), it is the method for utilizing molecular genetics that said inherited disease detects, and detects freeze-stored cell and whether has inherited disease.
According to the method for first aspect present invention, in step (8), it is to detect cell HLA-ABC/DR phenotype that said HLA-ABC/DR joins type.
According to the method for first aspect present invention, in step (9), said umbilical cord mesenchymal stem cells is frozen in liquid nitrogen through the programmed cooling process.
According to the method for first aspect present invention, in step (9), said umbilical cord mesenchymal stem cells is present in the cells frozen storing liquid.In one embodiment, this cells frozen storing liquid comprises DMEM-F12, DMSO 99.8MIN. and rHSA.In one embodiment, this cells frozen storing liquid comprises about 65 parts DMEM-F12, about 10 parts DMSO 99.8MIN., about 15 parts rHSA.In one embodiment, this cells frozen storing liquid comprises 50% low sugar DMEM nutrient solution, 40%FBS, 10% DMSO 99.8MIN..
According to the method for first aspect present invention, this method may further comprise the steps:
(1) sterilization and cleaning: in Biohazard Safety Equipment, the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off from the centre, be tiled on the aseptic 10cm cell cultures plate, utilize the PBS cleansing tissue, to reduce to organize top red corpuscle with alcohol;
(2) digestion process: the type i collagen enzyme of 0.1g is added the DMEM-F12 of 100ml, obtain Digestive system with the filtration of 20 μ m strainers then, the umbilical cord tissue that step (1) is obtained is transferred in another 10cm cell cultures plate, and umbilical cord tissue is cut into 1cm 3The tissue block of size; Tissue block is put in the Digestive system that has prepared; Digestion is 1.5 hours in 37 ℃ constant temperature shaking table, utilizes 100 μ m filter screens to remove remaining tissue block, and the volume ratio of pressing 1:1 adds mescenchymal stem cell substratum (15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to stop digestion; Centrifugal 10 minutes then, supernatant is removed and added behind the PBS re-suspended cell with rotating speed 1250rpm centrifugal 10 minutes again with rotating speed 1250rpm;
(3) preparation umbilical cord tissue frozen storing liquid: comprise the rHSA of 80 weight parts and the DMSO (DMSO 99.8MIN.) of 10 weight parts in the said umbilical cord tissue frozen storing liquid, the cold liquid storage for preparing is placed on 4 ℃ of refrigerators and preserves until use;
(4) cold the depositing of the full cell of umbilical cord: the frozen storing liquid that 2ml step (3) is obtained is added in the cell after the cleaning that step (2) obtains with re-suspended cell, extracts in a small amount that sample carries out cell counting, and re-suspended cell is managed 1ml with each then, cell density 1 * 10 6Every pipe adds in the frozen pipe, and this process needs under 4 ℃ coldcondition, to carry out, and frozen pipe is put in the programmed cooling box; First deepfreeze 0.5 hour under 4 ℃ temperature condition; Under the temperature condition again-80 ℃ freezing 1 day, frozen pipe is freezing in liquid nitrogen, subsequent use then.
Further, can comprise and the matching used method for resuscitation of frozen method:
(5) the full cell recovery of freezing and storing umbilical: the full cell of step (4) refrigerated umbilical cord is taken out from liquid nitrogen; Being placed on thaws in the water bath with thermostatic control begins to melt to half frozen storing liquid, utilizes mescenchymal stem cell substratum (it for example comprises 15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to carry out topical application and cleans the full cell of umbilical cord.
Further, can comprise that recovery back mescenchymal stem cell separates and the method for amplification:
(6) cell cultures: the cell 1ml that step (5) is obtained adds the mescenchymal stem cell substratum of 6ml, transfers to the T25 culturing bottle, puts the T25 culturing bottle into CO again 2Concentration is to cultivate in 37 ℃ of incubators of 5%; When being cultured to the 5th day the T25 culturing bottle is taken out from incubator, add 3ml mescenchymal stem cell substratum, continue to cultivate; In the time of the 9th day, the T25 culturing bottle is taken out from incubator; Carry out changing full the first time liquid, continue to cultivate, once changed liquid in per backward 2 days entirely;
(7) passage: the attached cell fusion rate when T25 culturing bottle the inside reaches about 80%; Digestive ferment capable of using (TrypLE Express) breaks away from T25 culturing bottle bottom with attached cell; Remove supernatant after centrifugal; And add mescenchymal stem cell substratum suspension cell again, be inoculated in the T25 Tissue Culture Flask and go down to posterity, and carry out amplification cultivation; After this changed liquid in per two days once after fusion rate reaches 80%, promptly get, go down to posterity again in case of necessity.
Further, can be directed against above step (8) gained umbilical cord mesenchymal stem cells, detect at least one item of following project: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type.
Further, the umbilical cord mesenchymal stem cells after can above step (8) gained being gone down to posterity is frozen in liquid nitrogen, subsequent use.
Further; Can be with the frozen and relevant fetus information of record in liquid nitrogen of the cell after going down to posterity; And the biological characteristics and the multidirectional differentiation potential that carry out cell are identified; And pair cell carries out molecular genetics diagnosis, preserves all related datas of cell, sets up the DB of umbilical cord stem cell and carries out related with freeze-stored cell.Therefore among the present invention in one aspect; The method of setting up umbilical cord stem cell DB is provided; It comprises that first aspect present invention is separated and the step of amplification umbilical cord mesenchymal stem cells; And following steps: the cell after will going down to posterity is the frozen and relevant fetus information of record in liquid nitrogen, and carry out the biological characteristics and the evaluation of multidirectional differentiation potential of cell, and pair cell carries out the molecular genetics diagnosis; Preserve all related datas of cell, set up the DB of umbilical cord stem cell and carry out related with freeze-stored cell.
In addition, in first aspect of the present invention, the method that the full cell cryopreservation of umbilical cord, recovery and recovery back stem cell are separated and increase is provided.Therefore second aspect present invention provides a kind of umbilical cord mesenchymal stem cells.
According to the umbilical cord mesenchymal stem cells of second aspect present invention, it obtains according to the said method of the arbitrary embodiment of first aspect present invention.
According to the umbilical cord mesenchymal stem cells of second aspect present invention, its cell purity is greater than 90%, for example greater than 95%.In one embodiment, said umbilical cord mesenchymal stem cells is after going down to posterity more than 3 generations, and cell purity is greater than 90%, for example greater than 95%.
Be further described in the face of the present invention down.The document that the present invention quoted, and the document of being quoted in the document, their full content is incorporated this paper by reference into.
In the present invention; In arbitrary technical scheme of the arbitrary aspect of the present invention; Its arbitrary technical characterictic is equally applicable to arbitrary embodiment of arbitrary aspect of the present invention, as long as they can not cause contradiction, and this being useful in each other in case of necessity can be done suitable modification.
In the present invention, term " umbilical cord mesenchymal stem cells " is meant the mescenchymal stem cell that derives from umbilical cord.Therefore in the present invention, particularly relate in the linguistic context of the present invention, term " umbilical cord mesenchymal stem cells " can exchange with " umbilical cord stem cell ", " stem cell ", " mescenchymal stem cell " and use, and indicates only if having clearly in addition.
In the present invention, term " PBS damping fluid " perhaps " PBS " be meant phosphate buffered saline buffer.Generality prescription and compound method and their general aspects that those skilled in the art know the PBS that under situation of the present invention, uses be pH value or pH scope for example.
In the present invention, term " umbilical cord " is meant neonatal umbilical cord, is meant the umbilical cord within 4 hours postpartum especially.
Mescenchymal stem cell (mesenchymal stem cell; MSC) for example human mescenchymal stem cell is separated from marrow the earliest; Derive from mesoblastic one type of tissue stem cell with multidirectional differentiation potential and self ability; Has ability (Caplan AI.Mesenchymal stem cells.J Orthop Res.1991 in vivo with under the external specified conditions to multiple adult cytodifferentiation such as scleroblast, chondrocyte, adipocyte, endotheliocyte, neurocyte, myocyte, liver cells; 9:641-650.Pittenger MF; Mackay AM, Beck SC, et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999; 284:143-147).Up-to-date research shows that mescenchymal stem cell has immunomodulatory and hematopoiesis support effect, and is easy to foreign gene importing expression.Therefore the seed cell of mescenchymal stem cell during still tissue-engineered bone, cartilage and cardiac muscle make up; Important carrier cell in the gene therapy; And, in HSCT and organ transplantation, be with a wide range of applications because mescenchymal stem cell promotes hematopoietic reconstitution and the anti-host response function of inhibition of transplant.Mescenchymal stem cell has the characteristic of external adherent growth, utilizes this specific character, people successfully from multiple tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood separation and Culture go out mescenchymal stem cell.
The mescenchymal stem cell of being reported at present is mainly derived from marrow, adopts density gradient centrifugation to obtain.Though separation method is easy, donor is got marrow need experience a relatively more painful operation, and has very high infection chance in the process of drawing materials and after drawing materials; Because the content of MSC is extremely rare among the human bone marrow, per 10 5～10 6Approximately have only 1 in the individual mononuclearcell, and along with the increase at age, the quantity of mescenchymal stem cell, propagation and differentiation capability descend significantly all in the marrow, make it in research with use in the especially clinical application and be restricted.Originate from the umbilical cord of embryonic development period extraembryonic mesoderm and form, contain a large amount of mesenchyme compositions by a matter, blood vessel and nurse cell.
Up-to-date research shows and contains abundant stem cell in the umbilical cord, and separation and Culture goes out these multipotential stem cells and will open up a brand-new and abundant source for experimental study and clinical application from umbilical cord.
Thereby existing separate stem cells is set up the method for stem cell bank many shortcomings are arranged still, for example purity is not enough and/or quantity is not high, and then demonstrates the expectation that these methods still can not satisfy people.The for example invention of CN 101270349A (one Chinese patent application numbers 200810061267.6, open day on September 24th, 2008) disclosed being entitled as " placenta mesenchyma stem cell separates and the amplification in vitro cultural method "; The invention of CN 101693884A (one Chinese patent application numbers 200910117522.9, open day on April 14th, 2010) disclosed being entitled as method of separating and extracting stem cells " a kind of from placenta, umbilical cord or fatty tissue "; CN 102146359A (one Chinese patent application numbers 201110005964.1, open day on August 10th, 2011) disclosed being entitled as " extracted the method for primary mesenchymal stem cells and serum-free amplification " from placenta invention.These methods are further improved remaining aspect the purity of extract and/or the recovery.
Because contain abundant hemopoietic stem cell in the bleeding of the umbilicus, people set up unbilical blood bank these important Biological resources of umbilical hemopoietic stem cell are stored, for multiple disease in the blood system and disease of immune system provide a kind of treatment means.Same umbilical cord mesenchymal stem cells is as a kind of more importantly stem cell resource; We use conventional cell cryopreservation method it to be chilled in-196 degrees centigrade medium-term and long-term preservation of profound hypothermia liquid nitrogen; Set up the umbilical cord stem cell bank, for stem-cell therapy is in the future preserved seed.
According to the method for the invention, the prescription of umbilical cord tissue frozen storing liquid and sample refrigerated flow process can successfully and effectively be protected in the refrigerated process the full cell of umbilical cord.According to the method for the invention; Utilize topical application substratum to be added the full cell of freezing and storing umbilical of recovery; Not only can the DMSO in the full cell be cleaned out; Can also avoid the use of the centrifugal step, thereby avoid the loss of cell in centrifugal process, and then reach the effect of the survival rate of effective raising recovery cell.According to the method for the invention, wherein the mescenchymal stem cell culture medium prescription can successfully and effectively carry out amplification in vitro to umbilical cord mesenchymal stem cells.The setting of according to the method for the invention, wherein changing the liquid time has been shortened attached cell and has been reached the time of specifying fusion rate.
The present invention is simple to operate; Convenient and practical; Can effectively protect the full cell of frozen umbilical cord, avoid cell survival rate loss in the recovery process, can obtain a large amount of mescenchymal stem cells; The differentiation performance is good, has the ability to cytodifferentiation such as scleroblast, adipocyte, chondrocyte, endotheliocyte, neurocyte.Comparison with existing method: MSC mainly adopts modus operandi extraction donor marrow or perfusion method to separate umbilical cord at present, and adherent culture obtains.It is few that this method is got cell quantity, and donor is being got the possibility that infection is all arranged after marrow is got in the marrow neutralization.The present invention's success separates the higher mescenchymal stem cell of a large amount of purity of acquisition in umbilical cord, and uses this method to set up the umbilical cord stem cell bank and lay in this stem cell that has application prospect.This method is simple and easy to do, and because umbilical cord is the same with bleeding of the umbilicus, the cell composition is inmature, and wide material sources conveniently are easy to get, and therefore method of the present invention will have broad application prospect in the clinical application of stem cell.
Embodiment
Can further describe the present invention through following embodiment, yet scope of the present invention is not limited to following embodiment.One of skill in the art can understand, and under the prerequisite that does not deviate from the spirit and scope of the present invention, can carry out various variations and modification to the present invention.The present invention carries out generality and/or concrete description to the material and the TP that are used in the test.Though for realizing that employed many materials of the object of the invention and working method are well known in the art, the present invention still does detailed as far as possible description at this.
The side that embodiment 1, the full cell cryopreservation of umbilical cord, recovery and recovery back stem cell separate and increase Method
The frozen method of umbilical cord tissue may further comprise the steps:
(1) sterilization and cleaning: in Biohazard Safety Equipment, the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off from the centre, be tiled on the aseptic 10cm cell cultures plate, utilize the PBS cleansing tissue, to reduce to organize top red corpuscle with alcohol;
(2) digestion process: the type i collagen enzyme of 0.1g is added the DMEM-F12 of 100ml, obtain Digestive system with the filtration of 20 μ m strainers then, the umbilical cord tissue that step (1) is obtained is transferred in another 10cm cell cultures plate, and umbilical cord tissue is cut into 1cm 3The tissue block of size; Tissue block is put in the Digestive system that has prepared; Digestion is 1.5 hours in 37 ℃ constant temperature shaking table, utilizes 100 μ m filter screens to remove remaining tissue block, adds mescenchymal stem cell substratum (wherein comprising 15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) by the volume ratio of 1:1 and digests stopping; Centrifugal 10 minutes then, supernatant is removed and added behind the PBS re-suspended cell with rotating speed 1250rpm centrifugal 10 minutes again with rotating speed 1250rpm;
(3) preparation umbilical cord tissue frozen storing liquid: comprise the rHSA of 80 weight parts and the DMSO (DMSO 99.8MIN.) of 10 weight parts in the said umbilical cord tissue frozen storing liquid, the cold liquid storage for preparing is placed on 4 ℃ of refrigerators and preserves until use;
(4) cold the depositing of the full cell of umbilical cord: the frozen storing liquid that 2ml step (3) is obtained is added in the cell after the cleaning that step (2) obtains with re-suspended cell, extracts in a small amount that sample carries out cell counting, and re-suspended cell is managed 1ml with each then, cell density 1 * 10 6Every pipe adds in the frozen pipe, and this process needs under 4 ℃ coldcondition, to carry out, and frozen pipe is put in the programmed cooling box; First deepfreeze 0.5 hour under 4 ℃ temperature condition; Under the temperature condition again-80 ℃ freezing 1 day, frozen pipe is freezing in liquid nitrogen, subsequent use then.
May further comprise the steps with the matching used method for resuscitation of frozen method:
(5) the full cell recovery of freezing and storing umbilical: the full cell of step (4) refrigerated umbilical cord is taken out from liquid nitrogen; Being placed on thaws in the water bath with thermostatic control begins to melt to half frozen storing liquid, utilizes mescenchymal stem cell substratum (wherein comprising 15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to carry out topical application and cleans the full cell of umbilical cord.
Recovery back mescenchymal stem cell separates and the method for amplification may further comprise the steps:
(6) cell cultures: the cell suspension 6ml that step (5) is obtained adds the mescenchymal stem cell substratum of 6ml, transfers to the T25 culturing bottle, puts the T25 culturing bottle into CO again 2Concentration is to cultivate in 37 ℃ of incubators of 5%; When being cultured to the 5th day the T25 culturing bottle is taken out from incubator, add 3ml mescenchymal stem cell substratum, continue to cultivate; In the time of the 9th day, the T25 culturing bottle is taken out from incubator; Carry out changing full the first time liquid, continue to cultivate, once changed liquid in per backward 2 days entirely;
(7) passage: the attached cell fusion rate when T25 culturing bottle the inside reaches about 80%; Digestive ferment capable of using (TrypLE Express) breaks away from T25 culturing bottle bottom with attached cell; Remove supernatant after centrifugal; And add mescenchymal stem cell substratum suspension cell again, be inoculated in the T25 Tissue Culture Flask and go down to posterity, and carry out amplification cultivation; After this changed liquid and once after fusion rate reaches 80%, go down to posterity again in per two days.
In the test of above step, the full cell of the umbilical cord after the recovery began to have attached cell to occur at the 13rd day that cultivates, and was cultured to the 23rd day cell confluency and reached 80%, and after 3 generations went down to posterity, cell purity was greater than 90%.
In the test of above step; Comprise the rHSA of 80 weight parts and the DMSO (DMSO 99.8MIN.) of 10 weight parts in the umbilical cord tissue frozen storing liquid; In two kinds of components of this frozen storing liquid; Any one ratio is departing from said ratio 20% when above, and frozen storing liquid can not effectively be protected umbilical cord tissue in the refrigerated process, is embodied in the isolating mescenchymal stem cell counting in freezing and storing umbilical tissue recovery back and obviously descends.
In the test of above step; Sample refrigerated flow process is a deepfreeze 0.5 hour under 4 ℃ temperature condition; Under the temperature condition again-80 ℃ freezing 1 day, the arbitrary temp of this freezing flow process was departing from said temperature 60% when above, and the time is departing from 80% when above; Umbilical cord tissue is not effectively protected in the refrigerating process, is embodied in the isolating mescenchymal stem cell counting in freezing and storing umbilical tissue recovery back and obviously descends.
In the test of above step, comprise 15 weight part FBS, 1 weight part L-Glutamine, 0.05 weight part Gentamicin and 84 weight part DMEM-F12 in the mescenchymal stem cell substratum.In four kinds of components of this substratum, during wherein any three kinds of fixed ratio, alternative ratio does not all reach 75% the 17th day fusion rate after departing from said ratio 10% when above, being passaged to the T25 culturing bottle.For example in the mescenchymal stem cell substratum that uses, comprise 1 weight part L-Glutamine, 0.05 weight part Gentamicin and 84 weight part DMEM-F12; And when 12 weight part FBS, 13.5 weight part FBS, 16.5 weight part FBS or 18 weight part FBS; Under four kinds of situation; After being passaged to the T25 culturing bottle, the 17th day fusion rate all between 53-74%.
In the test of above step, change the liquid time and be the 9th day of cell cultures and change liquid for the first time entirely, backward for once changing liquid in per 2 days entirely, the liquid time of changing is arbitrarily wherein being departed from above-mentioned time 10% when above, and attached cell did not reach fusion rate 80% in 20 days.For example change the liquid time and be and changed liquid entirely for the first time in the 12nd day of cell cultures, or once changed liquid entirely for per 4 days backward, under two kinds of situation, attached cell reached 80% at 22-25 days
In the test of above step, Digestive system is the DMEM-F12 that the type i collagen enzyme of 0.1g is added 100ml, and digestion time is 1.5 hours.Type i collagen enzyme content departs from above-mentioned content 10% when above in nutrient solution, or departs from above-mentioned time 10% when above in digestion time, and whether the full cell in the tissue can not high efficiency separation, separate and efficiently judge according to extracting the cell counting of sample in a small amount.
The side that embodiment 2, the full cell cryopreservation of umbilical cord, recovery and recovery back stem cell separate and increase Method
The method of reference implementation example 1 is carried out.The full cell of umbilical cord after the recovery began to have attached cell to occur at the 14th day that cultivates, and was cultured to the 24th day cell confluency and reached 80%.After 3 generations went down to posterity, cell purity was greater than 90%.
The side that embodiment 3, the full cell cryopreservation of umbilical cord, recovery and recovery back stem cell separate and increase Method
The method of reference implementation example 1 is carried out.The full cell of umbilical cord after the recovery began to have attached cell to occur at the 13rd day that cultivates, and was cultured to the 24th day cell confluency and reached 80%.After 3 generations went down to posterity, cell purity was greater than 85%.
The side that embodiment 4, the full cell cryopreservation of umbilical cord, recovery and recovery back stem cell separate and increase Method
The method of reference implementation example 1 is carried out.The full cell of umbilical cord after the recovery began to have attached cell to occur at the 12nd day that cultivates, and was cultured to the 23rd day cell confluency and reached 80%.
The side that embodiment 5, the full cell cryopreservation of umbilical cord, recovery and recovery back stem cell separate and increase Method
The method of reference implementation example 1 is carried out.The full cell of umbilical cord after the recovery began to have attached cell to occur at the 14th day that cultivates, and was cultured to the 23rd day cell confluency and reached 80%.
The cultivation and frozen of going down to posterity of embodiment 6, umbilical cord MSC
The cell of each acquisition of embodiment 1-5 is digested, get 1 * 10 after the digestion 6Cell joins in the 1ml cells frozen storing liquid (containing 65 parts of DMEM-F12+10 part DMSO 99.8MIN.s+15 parts of rHSAs), and it is frozen to enter into liquid nitrogen container at last through programmed cooling.
The biological characteristics of embodiment 7, umbilical cord MSC is identified
1, cell growth and morphology characteristics thereof
Through the separation and Culture of embodiment 1 and embodiment 6, the cultivation of umbilical cord mononuclearcell can obviously be seen the fusiformis attached cell at microscopically after 72 hours, can form the turbine-like cell clone about 10 days, can form the adherent layer of about 80% fusions after the had digestive transfer culture.In the culturing process, find the relative homogeneous of this cellular form, rate of propagation is fast, and adherent speed is fast, is prone to be passaged to 5-15 generation by trysinization, and its form and growth characteristic also do not have obvious change.
2, flow cytometry identification of M SC surface marker
Get respectively the 3rd, 6,9,12,15 generation cell, the Flow cytometry cell surface marker dynamic observes the variation of cell surface marker in the culturing process.The digestion collecting cell gets 8 * 10 behind the counting 6Individual cell, packing 16 pipes; PBS washes once, the centrifugal 10min of 1500rpm; Abandon supernatant, residual 100～200 μ l, piping and druming mixing cell; Add CD45, CD105, HLA-ABC, HLA-DR, each 10 μ l of UEA-1 antibody of CD14, CD29, CD31, CD34, CD44, CD54, CD73, CD80, CD86, CD166 antibody and the FITC mark of PE mark, and establish a pipe and be blank; Under 4 ℃, lucifuge reaction 30min; PBS washes once, the centrifugal 10min of 1500rpm; Directly the cell of mark is abandoned supernatant, adds 200 μ l PBS piping and druming mixing cell, and 1% Paraformaldehyde 96 of 200 μ l is fixed, put 4 ℃ to be measured, the upflowing cell instrument detects in 3 days.
Flow cytometer detects the surface marker of cell, dynamic observes the cell in the 3rd, 6,9,12,15 generations, does not have obviously to change.Not expressing the hematopoietic cell surface marker is CD14, CD31, CD34 (HSPC and endotheliocyte are positive), CD45 (white corpuscle is positive), CD54 (ICAM-1), CD80 (B7-1), CD86 (B7-2), the lasting feminine gender of HLA-DR (MHC-II quasi-molecule); CD29 and CD44 (acceptor of scleroproein and transparency grease hydrochlorate, stroma cell is expressed), CD73 (being SH-3,4), CD105 (being SH-2), CD166 (mesenchymal cell expression), HLA-ABC (MHC-I quasi-molecule) and UEA-1 (surface marker of endotheliocyte) are continuously the positive.After going down to posterity more than 3 generations, the cellular constituent homogeneous, purity is more than 95%.
3, the cell cycle of Flow cytometry umbilical cord MSC
Cell grows to about 80% when merging, digestion collecting cell about 1 * 10 6Individual, PBS washes once, and the ethanol of adding 70% is fixed, and 4 ℃ to be measured.During detection, the centrifugal ethanol that goes is washed once with PBS more earlier, adds RNase I 500u, 37 ℃ of reaction 30min, and PBS washes once, adds propidium iodide (PI, final concentration 50 μ g/ml) 1ml, room temperature lucifuge reaction 20min, last machine testing cell DNA content.
Through measure the 3rd generation and the 6th generation cell dna content, cell cycle analysis, G 0/ G 1Phase, S phase and G 2M phase proportion is respectively 96.35%, 96.66%, 1.11%, 0.09% and 2.54%, 3.25%.The result shows that the cell of vitro culture has typical stem cells hyperplasia characteristics, promptly has only few cell to be in the active propagation phase (1.11%, 0.09%), and most cell is in quiescent stage (96.35%, 96.66%).
4, the drafting of umbilical cord MSC growth curve and the mensuration of logarithmic phase doubling time
The cell in vegetative period of taking the logarithm, the digestion counting is processed cell suspension (every hole inoculation 0.5ml in 2 * 104/ml), 24 orifice plates, 37 ℃, 5%CO with the LG-DMEM substratum of 10%FBS 2, cultivate under the saturated humidity.Get 3 multiple holes every day, trypan blue dyeing back living cell counting number, calculating mean value was observed 7 days continuously.With the incubation time is transverse axis, and cell count is the longitudinal axis, draws cell growth curve.Calculate cell in the doubling time of logarithmic phase with the Patterson formula, i.e. Td=Tlg2/Lg (Nt/No), Td: the doubling time (h), T: cell increases to Nt used time (h), N: cell count by No.
Cytometric result draws cell growth curve through every day, calculates the doubling time.Can find out that by cell growth curve cell was in exponential phase of growth at 2-4 days.According to formula calculate the 5th generation cell doubling time of exponential phase of growth in 18-30 hour scope.
5, the evaluation of the multidirectional differentiation potential of umbilical cord MSC
(1) osteogenic induction
Above MSC of 3 generations is by 1 * 10 5Six orifice plates are inoculated in/hole, are put in 37 ℃, 5%CO 2, under the saturated humidity, cultivate 24h in the MSC substratum after, use instead and contain 10% through the DMEM-HG of screening FBS and add DEXAMETHASONE BP98 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM, be put in 37 ℃, 5%CO 2, under saturated humidity, cultivate, amount was changed liquid in per 3 days half, coinduction 2-4 week.Alkaline phosphatase staining identifies that scleroblast forms, and Von Kossa dyeing identifies that the bone tubercle forms.
Containing 10% DMEM-HG through screening FBS; Adding DEXAMETHASONE BP98 0.1 μ M, ascorbyl phosphate 50 μ M, β-phospho-glycerol 10mM cultivated for 1 week; Cellular form takes place significantly to change, and becomes polygon by fusiform inoblast appearance, is similar to neuronal cell appearance; It is outstanding that the long filament shape appears in the cell periphery, and can extend towards periphery.Continue to cultivate 2 weeks above after, calcified plaque appears in the cell matrix, mineralizer engenders, and begins to form the little junction structure of multilayer, after cultivating for 4 weeks, visible obviously calcification tubercle.The alkaline phosphatase staining of 2 whens week is strong positive reaction, reaches more than 95%, and in addition the inductive control group is then most of not negative, only is shown as the weak positive less than 5%, shows that cell transforms to scleroblast.Von Kossa dyeing can be dyed black with sedimentary calcium in the bone tubercle, induces the visible a large amount of black bone tubercle of group, tangible three-dimensional arrangement is arranged, and control group does not all have positive reaction at any time.
(2) become fat to induce
Above MSC of 3 generations is by 1 * 10 5/ hole is inoculated in six orifice plates, is put in 37 ℃, 5%CO 2, under the saturated humidity, in the MSC substratum, cultivate 24h after, use instead and contain 10% high sugared DMEM, and add DEXAMETHASONE BP98 1 μ M, INDOMETHACIN BP99 60 μ M, IBMX 0.5mM, Regular Insulin 5 μ g/ml through screening FBS, be put in 37 ℃, 5%CO 2, cultivate under the saturated humidity, amount was changed liquid in per 3 days half, and in 2 weeks of coinduction, oil red dyeing identifies that fat drips formation.
Containing 10% DMEM-HG through screening FBS; Adding DEXAMETHASONE BP98 1 μ M, INDOMETHACIN BP99 200 μ M, IBMX 0.5mM, Regular Insulin 10 μ g/ml cultivated 3 days; Form promptly takes place and changes in cell, is shunk gradually by fusiform inoblast appearance to shorten, and 90% above cell becomes cube or polygon; Cultured continuously 7 days, there have small fat to ooze under the mirror in the visible cell to be existing, and along with the prolongation of incubation time, fat drips and increases gradually and merges, and when cultivating for 2 weeks, the agglomerating fat of visible fusion drips and is full of whole cell.The fat that produces in the oil red O stain visible cell is dyed redness by specificity.
(3) become chondrocyte induction
3 generations above cell, according to every pipe 2 * 10 5The cell branch installs to the 15ml polypropylene centrifuge tube; Low-speed centrifugal makes cell in test tube, form micelle; In containing the DMEM-HG of 2.5%FBS, add Regular Insulin, Transferrins,iron complexes, each 6.25 μ g/ml of Sodium Selenite, BSA 1.25 μ g/ml, Sodium.alpha.-ketopropionate 1mM/L; Xitix phosphoric acid 37.5 μ g/ml, TGF-β 150ng/ml is put in 37 ℃, 5%CO 2, cultivate under the saturated humidity, amount was changed liquid, 2 weeks of cultured continuously in per 3 days half.
After inducing for 2 weeks the cell micelle is broken up smear, the visible II Collagen Type VI of alcian blue (Alcian blue) dyeing forms extracellular matrix and is blue, and control group does not have indigo plant and dyes.
Through the detection of above a series of data targets, demonstrate and use the MSC that the inventive method separation obtains, have ability to scleroblast, adipocyte, chondrocyte's differentiation, confirm that the MSC that the inventive method obtains has the stem cell characteristic.
The foundation of embodiment 8, umbilical cord stem cell bank
1, the detection of cytoactive
Utilize the trypan blue staining to count the number of frozen front and back viable cell.
2, the detection of cell contamination
Utilize small amounts of cells to cultivate, detect cell and whether receive the pollution of fungi and bacterium.Utilize the etiology method, detect cell whether receive hepatitis B two double, third liver, AIDS, cytomegalovirus, Epstein-Barr virus and syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2Ab, CMV-IgM and EBV-IgA, TRUST and infect.
3, the detection of inherited disease
Utilize the method for molecular genetics, detect freeze-stored cell and whether have inherited disease.
4, HLA-ABC/DR joins type
Detect cell HLA-ABC/DR phenotype, and place on record.
5, the investigation in cell source
Record fetus and father and mother's thereof detail file, and place on record.
6, the foundation of umbilical cord stem cell DB
After preserving normal umbilical cord stem cell, set up the DB of umbilical cord stem cell, comprising the first five items data, and foundation and freeze-stored cell is related.

Claims (10)

1. handle the method for the full cell of umbilical cord, this method comprises:
(A) below the stripped full cell of flesh tissue of umbilical cord is separated and frozen step:
(1) sterilization and cleaning: with thimerosal (for example alcohol) the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off, tiling is cleaned umbilical cord tissue through damping fluid (for example PBS damping fluid), to reduce red corpuscle on the umbilical cord tissue;
(2) digestion process: the umbilical cord tissue that step (1) is obtained is cut into tissue block; Tissue block is put into digestive ferment solution (for example it comprises type i collagen enzyme, DMEM-F12 without limitation); Digestion process 0.5-3 hour (for example 1.5 hours); Filter and remove tissue block, add the mescenchymal stem cell substratum to stop digestion, the cell that then digestion is obtained carries out cell and cleans;
(3) preparation umbilical cord full cells frozen storing liquid: comprise rHSA and DMSO (DMSO 99.8MIN.) in the full cells frozen storing liquid of said umbilical cord, the cold liquid storage for preparing is placed on deepfreeze under the temperature condition of 1 ℃ to 7 ℃ (for example 4 ℃);
(4) cold the depositing of the full cell of umbilical cord: at 0-15 ℃ (for example 1 ℃ to 7 ℃; For example 4 ℃) low temperature environment under; With re-suspended cell, then re-suspended cell is added in the frozen container in the cell after the cleaning that the full cells frozen storing liquid adding step (2) that step (3) is obtained obtains, frozen container is put into the programmed cooling device; Earlier under the temperature condition of 1 ℃ to 7 ℃ (for example 4 ℃) deepfreeze 0.2-2 hour (for example 0.5 hour); Under the temperature condition of-10 ℃ to-150 ℃ (for example-80 ℃) freezing 0.25-3 days (for example 1 day), frozen container is freezing in liquid nitrogen, subsequent use then again;
The step of (B) the stripped full cell of flesh tissue of frozen umbilical cord being recovered:
(5) the full cell recovery of freezing and storing umbilical: the full cell of step (4) refrigerated umbilical cord is taken out from liquid nitrogen, and thawing to 20%-70% (for example 50%) frozen storing liquid begins to melt, and utilizes the mescenchymal stem cell substratum to clean cell, so that the full cell recovery of frozen umbilical cord;
(C) the full cell of umbilical cord to recovery below carries out the step that mescenchymal stem cell separates and increases:
(6) cell cultures: in the cell that step (5) obtains, add the mescenchymal stem cell substratum of an amount of (for example 1-6 times of cell suspension volume amount), put into culture vessel, culture vessel is put in the incubator cultivated again; Be cultured to 2-7 days and culture vessel taken out from incubator when (for example 3-6 days, for example the 4th day, for example the 5th day); Add an amount of (for example 0.2-2 times of enchylema volume) mescenchymal stem cell substratum; Continue to cultivate, when 8-11 days (for example the 9th day), culture vessel is taken out from incubator, carry out changing full the first time liquid; Continue to cultivate, (for example 2 days) were once changed liquid entirely in every backward 1-3 days;
(7) passage: after the attached cell fusion rate in the culture vessel reaches 40%-70% (for example 60%); Utilize digestive ferment (for example TrypLe Express) that attached cell is broken away from container bottom, centrifugal, take supernatant away; Add mesenchymal stem cells substratum suspension cell again; Be inoculated in that culture vessel goes down to posterity and carry out amplification cultivation, liquid was changed once in (for example per 2 days) in after this every 1-3 days, after fusion rate reaches 70-90% (for example 80%); Promptly get umbilical cord mesenchymal stem cells, go down to posterity in case of necessity;
And optional
(D) following one or more step:
(8) to being directed against step (7) gained umbilical cord mesenchymal stem cells, detect at least one item of following project: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type;
(9) umbilical cord mesenchymal stem cells after step (7) gained is gone down to posterity is freezing in liquid nitrogen, subsequent use.
2. comprise the rHSA of 80 weight parts and the DMSO of 10 weight parts according to the process of claim 1 wherein in the said umbilical cord tissue frozen storing liquid.
3. according to the process of claim 1 wherein that in the step (2), the time of digestion process is 1-2.5 hour.
4. according to the process of claim 1 wherein in the step (5), what utilize that the mescenchymal stem cell substratum cleans that the full cell of umbilical cord adopts is topical application.
5. comprise FBS, L-Glutamine, Gentamicin and DMEM-F12 according to the process of claim 1 wherein in the said mescenchymal stem cell substratum.
6. according to the process of claim 1 wherein:
The FBS that contains 10-20% in the said mescenchymal stem cell substratum;
The L-Glutamine that contains 0.5-2% in the said mescenchymal stem cell substratum;
The Gentamicin that contains 0.02-0.1% in the said mescenchymal stem cell substratum; And/or
The DMEM-F12 that contains 80-90% in the said mescenchymal stem cell substratum.
7. contain the Gentamicin of the L-Glutamine of the FBS of 15 weight parts of having an appointment, about 1 weight part, about 0.05 weight part and the DMEM-F12 of about 84 weight parts according to the process of claim 1 wherein in the said mescenchymal stem cell substratum.
8. according to the method for claim 1, this method may further comprise the steps:
(1) sterilization and cleaning: in Biohazard Safety Equipment, the umbilical cord tissue surface is carried out disinfection, umbilical cord is cut off from the centre, be tiled on the aseptic 10cm cell cultures plate, utilize the PBS cleansing tissue, to reduce to organize top red corpuscle with alcohol;
(2) digestion process: the type i collagen enzyme of 0.1g is added the DMEM-F12 of 100ml, obtain Digestive system with the filtration of 20 μ m strainers then, the umbilical cord tissue that step (1) is obtained is transferred in another 10cm cell cultures plate, and umbilical cord tissue is cut into 1cm 3The tissue block of size; Tissue block is put in the Digestive system that has prepared; Digestion is 1.5 hours in 37 ℃ constant temperature shaking table, utilizes 100 μ m filter screens to remove remaining tissue block, and the volume ratio of pressing 1:1 adds mescenchymal stem cell substratum (15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to stop digestion; Centrifugal 10 minutes then, supernatant is removed and added behind the PBS re-suspended cell with rotating speed 1250rpm centrifugal 10 minutes again with rotating speed 1250rpm;
(3) preparation umbilical cord tissue frozen storing liquid: comprise the rHSA of 80 weight parts and the DMSO (DMSO 99.8MIN.) of 10 weight parts in the said umbilical cord tissue frozen storing liquid, the cold liquid storage for preparing is placed on 4 ℃ of refrigerators and preserves until use;
(4) cold the depositing of the full cell of umbilical cord: the frozen storing liquid that 2ml step (3) is obtained is added in the cell after the cleaning that step (2) obtains with re-suspended cell, extracts in a small amount that sample carries out cell counting, and re-suspended cell is managed 1ml with each then, cell density 1 * 10 6Every pipe adds in the frozen pipe, and this process needs under 4 ℃ coldcondition, to carry out, and frozen pipe is put in the programmed cooling box; First deepfreeze 0.5 hour under 4 ℃ temperature condition; Under the temperature condition again-80 ℃ freezing 1 day, frozen pipe is freezing in liquid nitrogen, subsequent use then;
Can also select in the said frozen method and freezing matching used recovery step:
(5) the full cell recovery of freezing and storing umbilical: the full cell of step (4) refrigerated umbilical cord is taken out from liquid nitrogen; Being placed on thaws in the water bath with thermostatic control begins to melt to half frozen storing liquid, utilizes mescenchymal stem cell substratum (15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) to carry out topical application and cleans the full cell of umbilical cord;
Can also selecting in the said frozen method to recover, the back mescenchymal stem cell separates and the step of amplification:
(6) cell cultures: the cell that step (5) is obtained is added the mescenchymal stem cell substratum of 6ml, transfers to the T25 culturing bottle, and putting the T25 culturing bottle into CO2 concentration again is to cultivate in 37 ℃ of incubators of 5%; When being cultured to the 5th day the T25 culturing bottle is taken out from incubator, add 3ml mescenchymal stem cell substratum, continue to cultivate; In the time of the 9th day, the T25 culturing bottle is taken out from incubator; Carry out changing full the first time liquid, continue to cultivate, once changed liquid in per backward 2 days entirely;
(7) passage: the attached cell fusion rate when T25 culturing bottle the inside reaches about 80%; Digestive ferment capable of using (TrypLE Express) breaks away from T25 culturing bottle bottom with attached cell; Remove supernatant after centrifugal; And add mescenchymal stem cell substratum suspension cell again, be inoculated in the T25 Tissue Culture Flask and go down to posterity, and carry out amplification cultivation; After this changed liquid in per two days once after fusion rate reaches 80%, promptly get, go down to posterity again in case of necessity;
And optional following one or more steps:
(8) to being directed against step (7) gained umbilical cord mesenchymal stem cells, detect at least one item of following project: cytoactive, cell contamination, inherited disease, HLA-ABC/DR join type;
(9) umbilical cord mesenchymal stem cells after step (7) gained is gone down to posterity is freezing in liquid nitrogen, subsequent use.
9. set up the method for umbilical cord stem cell DB; It comprises the step of each said method of claim 1-8; And following steps: the cell after will going down to posterity is the frozen and relevant fetus information of record in liquid nitrogen, and carry out the biological characteristics and the evaluation of multidirectional differentiation potential of cell, and pair cell carries out the molecular genetics diagnosis; Preserve all related datas of cell, set up the DB of umbilical cord stem cell and carry out related with freeze-stored cell.
10. umbilical cord mesenchymal stem cells, it obtains according to each said method of claim 1-8.
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