CN105062965A - Three-dimensional culture method for mesenchymal stem cells - Google Patents
Three-dimensional culture method for mesenchymal stem cells Download PDFInfo
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- CN105062965A CN105062965A CN201510477593.5A CN201510477593A CN105062965A CN 105062965 A CN105062965 A CN 105062965A CN 201510477593 A CN201510477593 A CN 201510477593A CN 105062965 A CN105062965 A CN 105062965A
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Abstract
The invention relates to a three-dimensional culture method for mesenchymal stem cells. The method comprises the steps of: acquiring mesenchymal stem cells, mixing the mesenchymal stem cells with PM polypeptide hydrogel, and then conducting culture. The method provided by the invention utilizes the PM polypeptide hydrogel with a three-dimensional network structure to provide the mesenchymal stem cells an environment simulating cell growth in human body, supplies sufficient growth space and nutritional requirements for the mesenchymal stem cells, and is conducive to amplification of the mesenchymal stem cells and improvement of the mesenchymal stem cells' activity.
Description
Technical field
The present invention relates to tissue engineering technique field, particularly the three-dimensional culture method of mescenchymal stem cell.
Background technology
Mescenchymal stem cell is a kind of cell with multi-lineage potential, and it can directional induction be the various kinds of cell such as insulin-like cell, chondrocyte.And mesenchymal cell is present in human multiple tissue; especially the mescenchymal stem cell of umbilical cord, placenta, adipose tissue-derived; there are wide material sources, be easy to gather, without advantages such as ethics problems; mass-producing can be induced to differentiate into insulin-like cell, for clinical treatment diabetes provide new technical scheme.
Because mescenchymal stem cell has stronger adherent property, therefore, in two-dimensional culture system, mescenchymal stem cell is easily assembled agglomerating, is unfavorable for that cell proliferation is cultivated, and the problem such as cytoactive is lower, viable cell ratio is low.
Summary of the invention
Technical problem to be solved by this invention is, in two-dimensional culture system, there is the defects such as cytoactive is low, cultivation effect is poor for mescenchymal stem cell in prior art, a kind of three-dimensional culture method that can improve the mescenchymal stem cell of cell proliferation rate and viable cell ratio is provided.
The technical solution adopted for the present invention to solve the technical problems is: the three-dimensional culture method providing a kind of mescenchymal stem cell, comprises the following steps: obtain mescenchymal stem cell, cultivate after being mixed by mescenchymal stem cell with PM polypeptide hydrogel.
In the three-dimensional culture method of mescenchymal stem cell provided by the invention, described PM polypeptide hydrogel is dissolved in by PM polypeptide that deionized water obtains.
In the three-dimensional culture method of mescenchymal stem cell provided by the invention, in described PM polypeptide hydrogel, in described PM polypeptide hydrogel, the massfraction of PM polypeptide is 0.1%-0.75%.
In the three-dimensional culture method of mescenchymal stem cell provided by the invention, in described PM polypeptide hydrogel, before described PM polypeptide hydrogel mixes with mescenchymal stem cell, also comprise the step adopting nutrient solution repeatedly to infiltrate described PM polypeptide hydrogel.
In the three-dimensional culture method of mescenchymal stem cell provided by the invention, in described PM polypeptide hydrogel, nutrient solution or complete culture solution based on described nutrient solution.
In the three-dimensional culture method of mescenchymal stem cell provided by the invention, in described PM polypeptide hydrogel, it is 2-4 time that described PM polypeptide hydrogel infiltrates number of times.
In the three-dimensional culture method of mescenchymal stem cell provided by the invention, before described PM polypeptide hydrogel mixes with mescenchymal stem cell, also comprise the step adopting the resuspended mescenchymal stem cell of complete culture solution, and make the concentration of mescenchymal stem cell be 1 × 10
4individual/mL-1 × 10
6individual/mL.
Implement the three-dimensional culture method of mescenchymal stem cell provided by the invention, following beneficial effect can be reached: utilize the environment that the PM polypeptide hydrogel with tridimensional network provides to simulate human inner cell and grow for mescenchymal stem cell, for mescenchymal stem cell provides sufficient growing space and nutritional needs, be conducive to the amplification of mescenchymal stem cell and improve the cytoactive of mescenchymal stem cell.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 is in test experience two of the present invention, adopts mescenchymal stem cell that 0%PM polypeptide hydrogel is cultivated before cultivation, electron microscopic observation arrive cell growth condition figure;
Fig. 2 is in test experience two of the present invention, after the mescenchymal stem cell adopting 0%PM polypeptide hydrogel to cultivate cultivates 12 days, electron microscopic observation arrive cell growth condition figure;
Fig. 3 is in test experience two of the present invention, adopts mescenchymal stem cell that 0.25%PM polypeptide hydrogel is cultivated before cultivation, electron microscopic observation arrive cell growth condition figure;
Fig. 4 is in test experience two of the present invention, the mescenchymal stem cell adopting 0.25%PM polypeptide hydrogel to cultivate in cultivation after 12 days, electron microscopic observation arrive cell growth condition figure;
Fig. 5 is in test experience two of the present invention, adopts mescenchymal stem cell that 0.5%PM polypeptide hydrogel is cultivated before cultivation, electron microscopic observation arrive cell growth condition figure;
Fig. 6 is in test experience two of the present invention, after the mescenchymal stem cell adopting 0.5%PM polypeptide hydrogel to cultivate cultivates 12 days, electron microscopic observation arrive cell growth condition figure;
Fig. 7 is in test experience two of the present invention, adopts mescenchymal stem cell that 0.75%PM polypeptide hydrogel is cultivated before cultivation, electron microscopic observation arrive cell growth condition figure;
Fig. 8 is in test experience two of the present invention, after the mescenchymal stem cell adopting 0.75%PM polypeptide hydrogel to cultivate cultivates 12 days, electron microscopic observation arrive cell growth condition figure.
Embodiment
For mescenchymal stem cell in solution prior art is in two-dimensional culture system, mescenchymal stem cell expanding effect is not good, the not high defect of activity, innovative point of the present invention is the three-dimensional culture method providing a kind of mescenchymal stem cell, by adding the PM polypeptide hydrogel with tridimensional network in the medium, human inner cell's growing environment can be simulated, for mescenchymal stem cell provides sufficient growing space and nutritional needs, thus achieve the object improving mescenchymal stem cell activity and amplification times.
The three-dimensional culture method of a kind of mescenchymal stem cell provided by the invention, comprises the following steps: obtain mescenchymal stem cell, cultivate after being mixed by mescenchymal stem cell with PM polypeptide hydrogel.
PM polypeptide hydrogel has tridimensional network, human inner cell's growing environment can be simulated, increase the contact area of mescenchymal stem cell and substratum and oxygen, meanwhile, because mescenchymal stem cell is stronger attached cell, the tridimensional network of PM polypeptide hydrogel, also for mescenchymal stem cell provides more sufficient growing space, the amplification of umbilical cord mesenchymal stem cells is more conducive to, simultaneously, PM polypeptide hydrogel water content is higher, also improves the activity of mescenchymal stem cell.
PM polypeptide hydrogel of the present invention, also known as PM gel peptide (PuraMatrix gel peptide), purchased from BD company; A kind of peptide that PuraMatrix polypeptide is made up of 16 amino-acid residues, do not comprise obvious somatomedin or cytokine, self-assembly forms nanofibrous structures, in water-soluble solution, hydrophobic and ion amino acid combines the gel forming tridimensional network, i.e. PM polypeptide hydrogel, can be used for the growing space that mescenchymal stem cell provides sufficient, expand the contact area of mescenchymal stem cell and nutrient solution, be conducive to the amplification of umbilical cord mesenchymal stem cells.Disclosed in current PM polypeptide hydrogel, purposes is for tissue regeneration, as bone filler, wound healing and for delivery system etc., and by after water-soluble for PM polypeptide in the present invention, utilize its tridimensional network to carry out the cultivation of mescenchymal stem cell, the culture effect of good mescenchymal stem cell can be obtained.
Particularly, the dimensional culture process of mescenchymal stem cell is:
1, deionized water dissolving PM polypeptide, obtains PM polypeptide hydrogel;
2, adopt basic culture solution repeatedly immersion step 1 obtain PM polypeptide hydrogel, infiltrate number of times be 2-4 time;
3, mescenchymal stem cell is obtained;
4, cultivate after the mescenchymal stem cell obtained in step 3 being mixed with the PM polypeptide hydrogel obtained in step 2.
In step 1, PM polypeptide is put into ultrasound bath pot sonic oscillation 30min, wherein, the water that this water-bath adopts is deionized water; If produce bubble in oscillatory process, be transferred to centrifuge tube centrifugal (4000 turns/5min) and remove.According to the final concentration of PM polypeptide in required PM polypeptide hydrogel, get EP pipe (the eppendorf pipe of 1.5mL, indicate the centrifuge tube of respective concentration), with the PM polypeptide hydrogel of deionized water dilution PM polypeptide stoste preparation desired concn, preferably, in PM polypeptide hydrogel, the massfraction of PM polypeptide is 0.1%-0.75%, PM polypeptide hydrogel concentration is excessive, then the reticulated structure of PM polypeptide hydrogel formation is too fine and close, be unfavorable for the transduction of signal between the transmission of cytokine and cell, or cause the anoxic of cell, be unfavorable for the multiplication culture of cell.
Because electrolyte substance can affect the solubleness of PM polypeptide hydrogel in water, therefore, the deionized water removing electrolyte substance is adopted to carry out dissolved dilution PM polypeptide hydrogel in this process.In addition, it is uneven that the existence of bubble makes PM polypeptide hydrogel disperse in deionized water, affect the amplification density of mescenchymal stem cell, and bubble structure is unstable, can break at any time, easily destroy the mescenchymal stem cell be attached on PM polypeptide hydrogel, therefore, in this process, avoid producing bubble as far as possible.
In step 2, the PM polypeptide hydrogel obtained in step 1 is inoculated in cell culture container, as in culture plate, disposablely inject respective concentration PM polypeptide hydrogel 50 μ L fast from culture hole center, avoid producing bubble as far as possible, if any bubble, puncture by fine needle, and ensure that high density PM polypeptide hydrogel is paved with at the bottom of hole, culture plate is placed in 37 DEG C of incubators and leaves standstill 15min.
Then, inject 200 μ L basic culture solutions along cultivation hole wall lentamente with 200 μ L liquid-transfering guns are careful, avoid producing bubble; Be placed in 37 DEG C of incubators standing promotion PM polypeptide hydrogel to solidify; Carefully inhale with liquid-transfering gun after 60min and abandon 150 μ L basic culture solutions, add the basic culture solution that 150 μ L are new, repeat this operation 2-4 time, every minor tick 60min, PM polypeptide hydrogel is infiltrated, to increase the cellular affinity on PM polypeptide hydrogel surface, is conducive to cell attachment, and make the cytokine in basic medium and nutritional factor part be dissolved in PM polypeptide hydrogel, maintain Growth of Cells; Last displacement 150 μ L complete culture solution also spends the night for subsequent use in 37 DEG C of incubators.
In step 3, the process obtaining mescenchymal stem cell is:
1) people's umbilical cord is obtained;
Take fully moon umbilical cord of Cesarean esction fetus about 4 ~ 6cm long, aseptically pump Cord blood, be placed in PBS damping fluid 4 DEG C preservation, process in 4h.
2) separation and purification mescenchymal stem cell
Long for about 4 ~ 6cm umbilical cord is put into super clean bench, and remove arteriovenous and adventitia, the PBS damping fluid of PH=7.4 carries out first rinsing 2-3 time, is cut into 1cm segment, then with PBS damping fluid repeatedly rinsing to remove blood as far as possible, be finally cut into meat gruel shape.
The umbilical cord of meat gruel shape is moved into 50mL centrifuge tube, add 5mL through 37 DEG C of preheatings, massfraction be 0.1% collagenase II, put into 37 DEG C of shaking baths vibration digestion 180min.Wherein, collagenase II is obtained by the PBS damping fluid of 0.1g collagenase digestion in 100ml.
Then, then add the DMEM/F12 of 30mL, repeatedly blow and beat, the sieved filter of cell, collect filtrate; Filtrate, with the centrifugal 10min of 1000 turns/min, is abandoned supernatant, is namely obtained mescenchymal stem cell.
3) Secondary Culture
By step 2) in the mescenchymal stem cell that is centrifugation down by 1 × 10
6the density of individual/mL is inoculated in culturing bottle with the DMEM/F12 containing 10%FBS (foetal calf serum), moves in incubator and cultivates.Next day, half amount changed liquid first, and every 3d changes liquid 1 time later.
After cell reaches fusion, prepare with trypsin trypsin solution: PBS damping fluid 200mL, trypsinase 0.5g, EDTA (ethylenediamine tetraacetic acid (EDTA)) 0.04g) incomplete digestion carries out Secondary Culture.
The mescenchymal stem cell propagation obtained due to P3 generation is more vigorous, and the cell obtained is more, and metabolism is more vigorous, and activity is comparatively strong, therefore, preferentially selects P3 to carry out Induction Transformation nesidioblast for mescenchymal stem cell in the present embodiment.
In step 4, the P3 getting acquisition in step 3, for mescenchymal stem cell, after tryptic digestion, is 1 × 10 with the resuspended rear adjustment cell density of complete culture solution
4-1 × 10
6individual/ml, adds 50 μ L mesenchyma stem cell suspensions containing in the culture plate of PM polypeptide hydrogel in step 2; Culture plate is placed in 5%CO
2, in 37 DEG C of incubators, change liquid every 2d.
For verifying that the three-dimensional culture method of mescenchymal stem cell provided by the invention has outstanding significance effect further, further describe below by way of specific experiment.
The propagation quantity of detection one, mescenchymal stem cell detects
1, according to table 1, prepare by deionized water dilution PM polypeptide stoste the PM polypeptide hydrogel that PM peptide masses mark is 0.75%, 0.5%, 0.25% and 0% respectively, the PM polypeptide hydrogel getting the above-mentioned concentration of 50 μ L is respectively inoculated in 96 orifice plates;
Table 1:
2, P3 is got for mescenchymal stem cell, with complete culture solution re-suspended cell liquid to 1 × 10
5individual/ml, respectively
In 96 orifice plates, every hole adds 50 μ L cell suspensions, then every hole is containing 5000 cells.Culture plate is placed in 5%CO
2, in 37 DEG C of incubators, change liquid every 2d.
3, get the enchylema cultivating 3d, 6d, 9d, 12d respectively and add CCK-8 solution 10 μ L, mix gently, put in incubator after hatching 4 hours, be determined at the absorbancy (OD value) at 450nm place by microplate reader.
Wherein, CCK-8 solution is: CellCountingKit is called for short CCK8 test kit, chemical name: 2-(2-methoxyl group-4-nitre phenyl)-3-(4-nitre phenyl)-5-(2,4-disulfobenzene)-2H-tetrazolium monosodium salt, be widely used in cell proliferation and Cytotoxic fast high-sensitive degree detection kit.
Detected result: as table 2, the mescenchymal stem cell quantity that cck-8 detection cytoactive result shows in each concentration PM polypeptide hydrogel all increases along with the time and increases, the strong and weak order of cell-proliferation activity is: 0.75%PM > 0.5%PM > 0.25%PM > 0%PM, the proliferation activity that the dimensional culture of visible mescenchymal stem cell in PM polypeptide hydrogel is cultivated than adherent two dimension is strong
Table 2:
| Number of days (d) | 0％PM | 0.25％PM | 0.5％PM | 0.75％PM |
| 1 | 0.51±0.01 | 0.53±0.02 | 0.55±0.01 | 0.56±0.03 |
| 2 | 0.65±0.02 | 0.68±0.01 | 0.74±0.02 | 0.81±0.02 |
| 3 | 1.11±0.02 | 1.22±0.02 | 1.51±0.03 | 1.76±0.01 |
| 4 | 1.62±0.01 | 0.18±0.01 | 1.77±0.03 | 1.94±0.02 |
| 5 | 1.91±0.02 | 2.52±0.03 | 1.90±0.02 | 2.37±0.03 |
| 6 | 2.23±0.01 | 3.09±0.02 | 2.23±0.02 | 2.57±0.01 |
| 7 | 2.24±0.02 | 3.10±0.03 | 2.33±0.02 | 2.68±0.03 |
Test experience two, the mescenchymal stem cell upgrowth situation in different concns PM:
As shown in figures 1-8, electric Microscopic observation finds mescenchymal stem cell adherent growth in 0%PM, in typical long shuttle-type; In 0.125%PM and 0.25%PM polypeptide hydrogel, part cell is had to sink to bottom adherent growth, part then dense growth in PM polypeptide hydrogel; In 0.5% with 0.75%PM, all in PM polypeptide hydrogel, there is agglomerating growth in mescenchymal stem cell, illustrate thus, the mescenchymal stem cell showed increased that the three-dimensional culture method of mescenchymal stem cell provided by the invention obtains, and PM polypeptide hydrogel massfraction is when being 0%-0.75%, PM polypeptide hydrogel concentration is larger, and mescenchymal stem cell proliferative amount is larger.
In sum, the three-dimensional culture method of mescenchymal stem cell provided by the invention, not only flow process is simple to operation, and adopt the PM polypeptide hydrogel with tridimensional network, for cell provides a three dimensional growth space, can the growing environment of analog cell in human body, mescenchymal stem cell for adherent growth provides sufficient growing space, and increase the contact area of mescenchymal stem cell and nutrient solution and oxygen, the propagation of mescenchymal stem cell can not only be promoted, and contribute to the activity improving mescenchymal stem cell.
Below by reference to the accompanying drawings embodiments of the invention are described; but the present invention is not limited to above-mentioned embodiment; above-mentioned embodiment is only schematic; instead of it is restrictive; those of ordinary skill in the art is under enlightenment of the present invention; do not departing under the ambit that present inventive concept and claim protect, also can make a lot of form, these all belong within protection of the present invention.
Claims (7)
1. a three-dimensional culture method for mescenchymal stem cell, is characterized in that: comprise the following steps: obtain mescenchymal stem cell, cultivate after being mixed by mescenchymal stem cell with PM polypeptide hydrogel.
2. the three-dimensional culture method of mescenchymal stem cell according to claim 1, is characterized in that, described PM polypeptide hydrogel is dissolved in deionized water by PM polypeptide and obtains.
3. the three-dimensional culture method of mescenchymal stem cell according to claim 2, is characterized in that, in described PM polypeptide hydrogel, the massfraction of PM polypeptide is 0.1%-0.75%.
4. the three-dimensional culture method of mescenchymal stem cell according to claim 1, is characterized in that, before described PM polypeptide hydrogel mixes with mescenchymal stem cell, also comprises the step adopting nutrient solution to infiltrate described PM polypeptide hydrogel.
5. the three-dimensional culture method of mescenchymal stem cell according to claim 4, is characterized in that, nutrient solution or complete culture solution based on described nutrient solution.
6. the three-dimensional culture method of mescenchymal stem cell according to claim 4, is characterized in that, it is 2-4 time that described PM polypeptide hydrogel infiltrates number of times.
7. the three-dimensional culture method of mescenchymal stem cell according to claim 1, it is characterized in that, before described PM polypeptide hydrogel mixes with mescenchymal stem cell, also comprise the step adopting the resuspended mescenchymal stem cell of complete culture solution, and make the concentration of mescenchymal stem cell be 1 × 10
4individual/mL-1 × 10
6individual/mL.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112300904A (en) * | 2020-10-27 | 2021-02-02 | 湖北省银丰鼎诚生物工程有限公司 | Graphene oxide hydrogel attached stem cell soaking device |
| CN113462644A (en) * | 2021-08-26 | 2021-10-01 | 吉林大学第一医院 | Method for efficiently culturing adipose-derived stem cells in 3D mode |
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| CN113462644A (en) * | 2021-08-26 | 2021-10-01 | 吉林大学第一医院 | Method for efficiently culturing adipose-derived stem cells in 3D mode |
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