CN105112360A - Mass culture method of umbilical cord mesenchymal stem cells - Google Patents
Mass culture method of umbilical cord mesenchymal stem cells Download PDFInfo
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- CN105112360A CN105112360A CN201510423138.7A CN201510423138A CN105112360A CN 105112360 A CN105112360 A CN 105112360A CN 201510423138 A CN201510423138 A CN 201510423138A CN 105112360 A CN105112360 A CN 105112360A
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Abstract
The invention provides a mass culture method of umbilical cord mesenchymal stem cells. The method comprises the following steps: obtaining umbilical cord Wharton's jelly; carrying out P0 generation culture on the umbilical cord Wharton's jelly until the cell fusion degree is 40-50%, and carrying out P1 subculture until the cell fusion degree is 85-95%; carrying out inoculating culture on P1 generation cells in a plate medium, and separating suspension cells non-adhered to the plate medium; and carrying out P2 subculture on the suspension cells in a medium containing a leukemia inhibition factor and a fibroblast-like growth factor. The above tissue adherent culture method allows adherence screening to be carried out through using a tissue culture plate after direct culture of P0 cells with low polytropy in early stage into P1 generation in order to maintain the uniformity of the cell differentiation degree; and the leukemia inhibition factor and the fibroblast-like growth factor are used to increase the adherence performance of the cells in the P2 generation culture process and inhibit differentiation, so the obtained umbilical cord mesenchymal stem cells have the advantages of low differentiation degree, trending to uniformity of the differentiation degree, strong adherence capability and collection facilitation.
Description
Technical field
The invention belongs to stem cell preparation field, be specifically related to a kind of mass propgation method of umbilical cord mesenchymal stem cells.
Background technology
Mescenchymal stem cell (mesenchymalstemcells, MSCs) has the potential of Multidirectional Differentiation under certain condition, is seed cell source important in Tissue Engineering Study.Owing to containing abundant mescenchymal stem cell in umbilical cord jelly of Wharton (or leading to glue also known as magnificent Tong Shi glue, Wal), so a large amount of preparations of mescenchymal stem cell adopt separation and Culture from umbilical cord to obtain more, and the method for preparation is generally tissue explants adherent method and enzyme digestion.Common enzyme digestion carries out mass propgation after adopting Collagenase and tryptic digestion umbilical cord jelly of Wharton tissue, in this method trypsinase be by dissociated cells between Saliva Orthana, glycoprotein and affect cytoskeleton thus make cellular segregation, very strong destruction is all had to the protein ingredient of intercellular substance and epicyte protein, therefore cause the stem cell be separated to sustain damage and cause activity not good, be difficult to amplify desired number and active mescenchymal stem cell.
Based on above-mentioned, when needing the umbilical cord mesenchymal stem cells of the low damage of a large amount of preparation, many employing tissue block adherent are cultivated and are carried out; But the existing common tissue block adherent method time is long, acquisition in early stage rate is not high, and Engineering operation amount is huge.Such as use be cultured to required P2 for time, a umbilical cord can reach more than 3,000,000,000 through cultivating the cell quantity obtained, the cell of such quantity needs the quantity of T175 culturing bottle to reach 500 more than, and because the ability of cell attachment is also incomplete, between being half adherent degree increase operation easier (than being easier to the loss that comes off), so the process that cell reclaims is great workload.And in the process of cultivating, because tissue block infiltration medium in early stage grows to the training method of cell spilling, to cultivate the splitting degree of the cell obtained all different with differentiation degree, easily obtain cell uniformity in the later stage not enough, cause tissue block adherent method mass propgation in use still to there is multiple restriction.
Summary of the invention
Object of the invention process is to overcome existingly organizes the adherent mesenchyme mass propgation that carries out to produce cell and there is differentiation heterogeneity and difficult defect of collecting, and provides a kind of mass propgation method of umbilical cord mesenchymal stem cells.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
A mass propgation method for umbilical cord mesenchymal stem cells, comprises the steps:
Obtain umbilical cord jelly of Wharton;
It is 40% ~ 50% that described umbilical cord jelly of Wharton is carried out P0 culture to cytogamy degree;
It is 85% ~ 95% that the cell of described P0 culture is passed P1 culture to cytogamy degree, and P1 is for cell for results;
Described P1 is carried out inoculation culture for cell in plate culture medium, is separated not by suspension cell that plate culture medium adheres to;
By described suspension cell in containing leukaemia inhibitory factor with become the substratum of fiber like growth factor to pass P2 culture.。
The above-mentioned method organizing adherent culture of the present invention, organize in original cultivation based on fresh jelly of Wharton, cell quality differentiation, variation and amplification ability are not high, the change of P0 cell generation polytropism itself is lower in early days, so after being directly cultured to P1 generation, the cell tissue culture plate in P1 generation is carried out adhesion screening, the cell P1 that acquisition is not broken up or differentiation degree is lower, for cell, keeps the homogeneous of cytodifferentiation degree; Afterwards by leukaemia inhibitory factor with become the fiber like growth factor adherent performance of increase cell in P2 culture and Inhibited differentiation, thus obtain degree of differentiation lower and reach unanimity, adherent ability is beneficial to more by force the umbilical cord mesenchymal stem cells of collection.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Example of the present invention proposes a kind of mass propgation method of umbilical cord mesenchymal stem cells, comprises the steps:
S10, obtains umbilical cord jelly of Wharton;
S20, is seeded to umbilical cord jelly of Wharton in culturing bottle that to carry out being cultured to cytogamy degree be 40% ~ 50%;
S30, step S20 cultured cells being carried out P1 Secondary Culture to cytogamy degree is 85% ~ 95%;
S40, carries out inoculation culture by the cell of the P1 Secondary Culture of step S30 in tissue culturing plate, is separated not by cell that tissue culturing plate adheres to;
S50, by step S40 obtain cell in containing leukaemia inhibitory factor and become fiber like growth factor substratum biography P2 generation.
The above-mentioned method organizing adherent culture of the present invention, organize in original cultivation based on fresh jelly of Wharton, cell quality differentiation, variation and amplification ability are not high, the change of P0 cell generation polytropism itself is lower in early days, so after being directly cultured to P1 generation, the cell tissue culture plate in P1 generation is carried out adhesion screening, the cell P1 that acquisition is not broken up or differentiation degree is lower, for cell, keeps the homogeneous of cytodifferentiation degree; Afterwards by leukaemia inhibitory factor with become the fiber like growth factor adherent performance of increase cell in P2 culture and Inhibited differentiation, thus obtain degree of differentiation lower and reach unanimity, adherent ability is beneficial to more by force the umbilical cord mesenchymal stem cells of collection.
And wherein in the above-mentioned embodiment of the present invention, P1 is carried out inoculation culture for cell tissue culturing plate by step S40, utilize cytodifferentiation to be because receive the orientation of environment or nondirectional induction, and in these processes, cell is induced or the communication that stimulates or contact, is completed by cell adhesion molecule.So cell adhesion molecule to participate between cell and cell and interactional molecule between cell and extracellular matrix, this material of cell that differentiation degree is higher is more, so the difference of the cell adhesion utilizing cell adhesion molecule to cause of tissue culturing plate in the present invention, noble cells to be attached in tissue culturing plate thus to remove noble cells.So the cell tentatively sieved through step S40, substantially be exactly the lower and more consistent mescenchymal stem cell of degree of differentiation, the level of the growth of cells intact can be promoted like this, because are cells that degree of differentiation is higher or division algebraically is higher when wherein there being some in cultured cells group, these cells can be stablized more than on other cell quality, and metabolism has inducing function; Such as divide the very high cell of number of times, when entering the paracme, its normal cell that also can affect other enters the paracme.
After further step S40, step S50 proceeds to pass P2 for mass propgation, adopts in this step and adds two kinds of cytokines in the medium; Wherein leukaemia inhibitory factor self is that a kind of Spontaneous Differentiation that suppresses is to maintain the lymphokine of embryonic stem cell long-term survival, adding in the medium can preliminary Inhibited differentiation, therefore can ensure in a large amount of succeeding generations of cell, do not occur individuality or local differentiation cataclysm situation, even if create differentiation also can maintain more homogeneous and lower level.
And further cell is originally in the process of mass propgation, organize cell in adherent method then adherent through of short duration suspension, length different latent period is spent subsequently under adhered state, enter the exponential phase of growth of a large amount of division, but cell will carry out growing and dividing under adhered state, adherent ability itself is also be not as strong as inoblast etc., and many places are in half adherent degree, so easily suspension later collection cell is also more difficult in cultivating.So also add into fiber like growth factor in the medium in the process of P2 culture, the stromal precursor cell PCNA that non-adherent grows can be promoted, thus promote adherent ability.
But, need the amount controlling above-mentioned cytokine further in the present invention, the change of fiber like growth factor to cell quality itself is become to be partial to proterties induction, the simple adherent ability promoting cell is only needed all can in the present invention, after a large amount of interpolation, cell may be directed induction to inoblast differentiation, so control 2 ~ 4 μ g/L in the invention process process.Can ensure simple to strengthen adherent performance under extremely low consumption, also can not be induced in a large number make cell proterties directed change to the degree of breaking up to fibrocyte.
Simultaneously, in the present invention leukaemia inhibitory factor with become the process of fiber like growth factor to be in the P2 culture stage to carry out, if carried out in the P1 stage, the multiplication capacity of early stage cell and activity are not strong, if carry out in early days at the proliferation activity causing cell shape change meeting T suppression cell in the process processed, and early stage process also may produce the possibility of Induction of committed differentiation.Carry out so the stage of process is placed in P2 generation by the present invention, now through the multiplication culture in P1 generation, the shape that the proliferation activity of cell has obtained activation and cell itself also tends towards stability, and also has lifting, can compare the period be suitable for as cytokine process for differentiation-inducing tolerance.
And in early days in order to the Character change postponed in the culturing process of cell own easily sends out the stage, and delay the division algebraically making cell, adopting umbilical cord jelly of Wharton inoculation culture to cytogamy degree in step S20 is 40% ~ 50%, instead of to be generally cultured to degrees of fusion be about 80%, be the stage being in separation and original cuiture in this stage, after in vitro, the ability of division growth own is also lower.So being now cultured to degrees of fusion is that about 80% required time is long, be directly once cultured to logarithmic phase peak quantity, the division algebraic sum differentiation degree difference of new old cell is larger; And a lot of cell meeting overgrowth, the natural differentiation rate reducing cell can be caused; So when to be first cultured to cytogamy degree in the present invention be 40% ~ 50%, change environment and carry out P1 and go down to posterity, now cell is cultivated through former P0; Tentatively there is the activity of reasonable propagation, made it again be in the mild phase after changing fresh growing environment, delay its aging degree.
Further, state on the invention in embodiment, when P1 passes P2 cell cultures for cell, preferably adopt the splitting ratio of 1:4 ~ 1:10 to go down to posterity.In force, keep the splitting ratio of cell very important, the best ratio of going down to posterity is 1:8, the natural differentiation of cell can be made to drop to minimum.And zooblast meeting in the process of cultivating is because the certain tendency of reuniting of adhesivity generation of own cells, much more slightly cell can be rolled into a ball with the palms loose separating than being easier to by the said amount adding substratum when going down to posterity.And if there is mescenchymal stem cell to reunite when going down to posterity, so can be spread out, can not obtain nutriment and growing space and dead with inner cell after avoiding reuniting, the cell of death can induce again other normal cells to start death; And the cell after a large amount of dispersion can also reduce the possibility of the natural differentiation of cell.
So, tendency of reuniting is there is based on above-mentioned mescenchymal stem cell, so after the present invention carries out digestion process with trypsinase-EDTA further before going down to posterity, by the tryptic digestion process cell with EDTA chelating, single to be separated between agglomerated or adhesion cell together, thus the natural differentiation of the Growth of Cells be beneficial in follow-up culturing process and minimizing cell.But because the injury of trypsinase in enzyme digestion-Collagenase associating cell membrane albumen is larger, easy destruction cell, so adopt EDTA chelating to modify in the present invention, and the mass percentage concentration controlling trypsinase-EDTA is 0.2 ~ 0.3% to digest as far as possible, control in this Valid concentration, avoid aggregation guaranteeing substantially to digest separation, also avoid cell membrane to damage as far as possible.
Meanwhile, in above-mentioned implementation process, all need in P2 culture to carry out changing liquid, be all adopt a kind of substratum to carry out in common situation and method, reduce the efficiency of propagation to avoid the cultivation renewed to need cell again to adapt to; Suppress the object of natural differentiation based in growth in the present invention, adopt and alternately change liquid containing the DMEM/F12 substratum of 8 ~ 12%FBS and these two kinds of substratum of Human umbilical cord mesenchymal stem cell culture medium; During after testing, early stage P0 cultivates, can not the too multiplication capacity of T suppression cell and activity, so of the present invention above-mentioned when alternately changing liquid, carry out in the culturing process in P2 generation, this phase cell itself has possessed reasonable multiple fission ability, so a little alternately carry out changing liquid with two kinds of nutrient solutions, also smaller on the impact of the speed of growth.
The understanding of those skilled in the art can be easier to for the ins and outs and process approach that make above-mentioned enforcement of the present invention and implement reference, highlight the quality of the umbilical cord mesenchymal stem cells that the present invention is prepared in a large number simultaneously, be illustrated below by way of specific embodiment.
Embodiment 1
S11, cleans 2 times by the umbilical cord physiological saline producing the 15cm length that Operation theatre obtains from hospital, really makes umbilical cord surface not containing bloodstain; Umbilical cord two ends are removed, cleans up the bloodstain of quiet artery in umbilical cord with physiological saline as far as possible;
S12 surgical scissors is cut into segment umbilical cord, every section of about 4cm, and removes vein blood vessel, arteries successively, isolates magnificent Tong Shi glue, puts into culture dish physiological saline and cleans;
S13, is cut into about 2 ~ 3mm by magnificent Tong Shi glue
3in small, broken bitsly organize fritter, granularity keeps homogeneous as far as possible.
S21, is inoculated into the magnificent Tong Shi glue shredded in T-75 culturing bottle, and does 5 and repeats contrast;
S22, after adding perfect medium, puts into incubator control condition 37 DEG C, 5%CO in each culturing bottle of step S21 by culturing bottle level
2cultivate;
Within after the primary separation and Culture of S23, step S22 the 5th day, carry out full dose and change liquid, remove all old nutrient solution, add fresh perfect medium; Within after full dose changes liquid the 7th day, carry out half amount and change liquid; After to carry out once half amount every three days and change liquid, until when the cytogamy degree in culturing bottle is 40%-50%, the cell carrying out step 30 passes P1 culture.
S31, to step S23 cultivate terminate culturing bottle in add mass percentage concentration be 0.25% trypsinase-EDTA digest, stop digestion after collected by centrifugation P0 for human umbilical cord mesenchymal stem cells.
S32, by the P0 of step S31 collected by centrifugation for the quantity of human umbilical cord mesenchymal stem cells according to T-75 culturing bottle during original cuiture, principle by one to one is inoculated in the T-175 culturing bottle of same quantity for cell collecting the P0 obtained, and is placed in incubator control condition 37 DEG C, 5%CO
2cultivate.
S41, collects P1 for cell when the cytogamy degree that step S32 is cultured in culturing bottle is 85% ~ 95%;
S42, the P1 collected by step S41 is seeded on tissue culture dishes (being solid ES cel culture plates substratum in ware) for cell, (noble cells will stick in this time in 2 hours to put into incubator, and mescenchymal stem cell cell will keep suspending), the suspension cell do not adhered to is poured out;
S51, the trypsinase-EDTA being 0.25% by the cell mass percentage concentration that step S42 is separated digests, and stops the rear centrifugal collecting cell of digestion;
S52, the cell of step S51 is passed P2 culture according to the ratio that goes down to posterity of 1:8, by the DMEM/F12 substratum (autogamy) of 8 ~ 12%FBS and Human umbilical cord mesenchymal stem cell culture medium (hundred En Wei biotech firm BW110013 models) in the process of cultivating, alternately change liquid; And control culture condition 37 DEG C, 5%CO
2cultivate;
S53, carry out when the cytogamy degree in culturing bottle is 85% ~ 95% P2 for cell harvesting and frozen P2 for human umbilical cord mesenchymal stem cells.
In order to verify actual significance of carrying out the effect of cultivating in above-mentioned implementation process, to the situation of its Character change of cell observation of P2 culture, it is 40.8 ± 3.69 hours that the cell wherein finding to observe whole culturing bottle urgees the adherent time, and the time comparing conventional organization adherent culture decreases about about 8h; The thinner slight full dose of making peace of action can be adopted after cell attachment to change liquid operation carry out simultaneously, and the drain cell of minute quantity only detected from substratum, the ability of cell attachment has lifting; Reduce the operation easier in culturing process, be more convenient to later collection.
The P2 of results is carried out Phenotypic examination for cell, and HLA-I quasi-molecule, mescenchymal stem cell surface common molecule CD105, CD73 of each group cell are all expressed as the positive; And HLA-II quasi-molecule and hemopoietic stem cell surface molecule CD34, LCA CD45 are all expressed as feminine gender.Illustrate that the type of cell is more consistent, more consistent in phenotype, hypotype is less, can think the differentiation of cell and homogeneous degree or reasonable.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (6)
1. a mass propgation method for umbilical cord mesenchymal stem cells, is characterized in that, comprise the steps:
Obtain umbilical cord jelly of Wharton;
It is 40% ~ 50% that described umbilical cord jelly of Wharton is carried out P0 culture to cytogamy degree;
It is 85% ~ 95% that the cell of described P0 culture is passed P1 culture to cytogamy degree, and P1 is for cell for results;
Described P1 is carried out inoculation culture for cell in plate culture medium, is separated not by suspension cell that plate culture medium adheres to;
By described suspension cell in containing leukaemia inhibitory factor with become the substratum of fiber like growth factor to pass P2 culture.
2. the mass propgation method of umbilical cord mesenchymal stem cells as claimed in claim 1, is characterized in that, by described P1 for cell before plate culture medium carries out inoculation culture, also comprise:
Be the trypsinase-EDTA digestion process processed of 0.2 ~ 0.3% for cell mass percentage concentration by described P1.
3. the mass propgation method of umbilical cord mesenchymal stem cells as claimed in claim 1 or 2, is characterized in that, is passed by described suspension cell in the process of P2 culture, becomes the concentration of fiber like growth factor to control 2 ~ 4 μ g/L in substratum.
4. the mass propgation method of umbilical cord mesenchymal stem cells as claimed in claim 1 or 2, it is characterized in that, described suspension cell is passed in the process of P2 culture, adopt and alternately change liquid containing the DMEM/F12 substratum of 8 ~ 12%FBS and Human umbilical cord mesenchymal stem cell culture medium.
5. the mass propgation method of umbilical cord mesenchymal stem cells as claimed in claim 1 or 2, is characterized in that, is passed by described suspension cell in the process of P2 culture, goes down to posterity according to the splitting ratio of 1:4 ~ 1:10.
6. the mass propgation method of umbilical cord mesenchymal stem cells as claimed in claim 1 or 2, it is characterized in that, obtain in described umbilical cord jelly of Wharton step, described umbilical cord jelly of Wharton is of a size of 2 ~ 3mm
3.
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| CN105477017A (en) * | 2015-12-03 | 2016-04-13 | 深圳爱生再生医学科技有限公司 | Mixed stem cell preparation for treating diabetic foot and preparation method of mixed stem cell preparation |
| CN114008189A (en) * | 2018-06-19 | 2022-02-01 | 加拿大干细胞科技公司 | Adaptive passage system, method and device for cell culture |
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| CN105477017A (en) * | 2015-12-03 | 2016-04-13 | 深圳爱生再生医学科技有限公司 | Mixed stem cell preparation for treating diabetic foot and preparation method of mixed stem cell preparation |
| CN114008189A (en) * | 2018-06-19 | 2022-02-01 | 加拿大干细胞科技公司 | Adaptive passage system, method and device for cell culture |
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