CN107099502A - A kind of nutrient solution - Google Patents
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Abstract
The invention belongs to stem cell field, more particularly to a kind of nutrient solution.The nutrient solution that the present invention is provided includes human chondrocytes culture supernatant and DMEM nutrient solutions, and wherein human chondrocytes culture supernatant contains cell factor and active ingredient of a variety of rush hUC MSCs to cartilage cell's specific differentiation.Test result indicates that, the nutrient solution provided using the present invention can effectively facilitate specific differentiations of the hUC MSCs to cartilage cell.
Description
Technical field
The present invention relates to stem cell field, more particularly to a kind of nutrient solution.
Background technology
With the development of the social economy, living standard and improve and aging population phenomenon, using cartilage surface destruction as
The incidence of disease of the diseases such as the osteoarthritis mainly showed increasingly increases.Therefore, how it is minimally invasive repair damage cartilage, alleviate patient
Pain, to improve the quality of living be exactly one of emphasis direction of Osteopathic Medicine area research for a long time.Osteoarthritis is orthopaedics
Clinical common disease, the articular cartilage defect caused due to retrogression pathological changes, wound etc., it has also become cause maimed, influence life
The major reason of bioplasm amount.Articular cartilage is a kind of vesselless tissue, and cartilage cell is mainly by knuckle synovia and cell peripheral
Matrix supply nutrition is energized with anerobic glycolysis, and the repair ability of its own is very limited.Articular cartilage will cause pass once damaging
Pain, unstable and stiff is saved, the ossified of joint can be accelerated, the speed of extracellular matrix degradation is more than the synthesis of new matrix, then lost
Permanent lesion is stayed, so as to cause arthritic generation.Traditional treatment method such as subchondral bone Drilling, cartilage transplantation, bone
Film or perichondrium transplanting, chondrocyte cell transplantation etc. lack the mechanics of normal hyaline because repair tissue is based on fibrocartilage
Performance and durability, therefore promising result can not be obtained.
Encouraging prospects are illustrated using Method of Tissue Engineering repairing articular cartilage defect.Seed cell is tissue work
The most important condition of journey cartilage formation.Mescenchymal stem cell (mesenchymal stem cells, MSCs) is dived more as a kind of
Energy stem cell, because it successfully avoids the problems such as derived from embryonic stem cells shortage, allosome repulsion, ethics, in recent years
Turn into the study hotspot of stem cell field.Its not only hematopoiesis support system, can also be to mesoderm and the tissue of ectodermal origin
Differentiation, can be divided under certain experiment condition control myocyte, Gegenbaur's cell, fat cell, nerve cell, liver cell,
Cardiac muscle cell etc..Therefore, MSCs has wide potential applicability in clinical practice, is cell replacement therapy and the preferred seed of organizational project
Cell, is the study hotspot of fields of implantation and Autoimmune disease treatment.
Recent study shows, MSCs is the pluripotent stem cell that a class derives from mesoderm, with self-renewing and many
To the ability of differentiation, the reparation and regeneration that can be damaged as seed cell promotion organization.Because its is tissue-derived extensively, it is easy to body
Outer amplification, the low advantage of immunogenicity, have been increasingly becoming organizational project and the optimal seed cell of cell transplantation.At present, bone
Marrow source MSCs is the most frequently used seed cell, but its quantity and differentiation potential are substantially reduced and reduced with advancing age,
And actual bodily harm easily is caused to donor when drawing materials.Therefore, the cell for being highly desirable to find new autologous and heteroplastic transplantation comes
Source.This new cell is except that must possess the characteristics of having stronger proliferative ability and multi-lineage potential than derived from bone marrow MSCs
Outside, it is necessary to materials facility, viral pollution is not easily caused, and do not limited by ethics.From Mitchell etc. first from navel
Umbilical cord mesenchymal stem cells (hUC-MSCs) are had successfully been isolated out in band and are confirmed after its many differentiation potential, and umbilical cord tissue is originated
The research of mescenchymal stem cell is attracted wide attention, and increasing research is focused in the field.Sent out from hUC-MSCs
So far now, Chinese and overseas scholars has carried out considerable research, inhales the features such as its low stain having, source sufficient, molecular marker for increased proliferation
Numerous researchers are drawn.The significance of separation and Extraction mescenchymal stem cell is from umbilical cord, and umbilical cord is discarded as childbirth
Thing, wide material sources, convenient material drawing is not limited by any ethics and legal principle.
The microenvironment that MSCs differentiation grows with it has substantial connection, therefore, and external evoked MSCs differentiation is by taking
The true environment and necessary condition of respective organization cell growth in different method analogue bodies.
Traditional treatment method such as subchondral bone Drilling, cartilage transplantation, periosteum or perichondrium transplanting, chondrocyte cell transplantation
Deng because repair tissue is based on fibrocartilage, lack the mechanical property and durability of normal hyaline, therefore satisfaction can not be obtained
Effect.Existing hUC-MSCs is not good into cartilage differentiation induction liquid effect, and the specificity of induction differentiation is not strong.
The content of the invention
In view of this, it is an object of the invention to provide a kind of nutrient solution, the culture liquid energy that the present invention is provided is significantly improved
Specific differentiation from hUC-MSCs to cartilage cell.
The invention provides a kind of nutrient solution, human chondrocytes culture supernatant and DMEM nutrient solutions are included.
It is preferred that, the human chondrocytes culture supernatant and the volume ratio of DMEM nutrient solutions are：0.5~1.5:1.5~
1.5。
It is preferred that, the human chondrocytes culture supernatant is prepared by the following：
A) articular cartilage is collected, cleaning is sequentially passed through, shreds, centrifuges, filtering, obtain cell suspension；
B) it will be cultivated after cell suspension inoculation, during cell fusion to covering culture dish bottom more than 80%, digested,
Passage, obtains supernatant crude product；
C) supernatant crude product is centrifuged, takes supernatant to be filtered, microscopy, obtain the human chondrocytes culture supernatant
Liquid.
It is preferred that, the screen cloth of the step a) filterings is 180~220 mesh.
It is preferred that, the cell density of the step b) inoculations is 0.5~1.5 × 109/L。
It is preferred that, the filter particle diameter of the step c) filterings is 0.2~0.24 μm.
It is preferred that, the DMEM nutrient solutions include TGF, dexamethasone, L-AA, ITS Premix
And FBS.
It is preferred that, the TGF is any one in β 1 or β 2.
It is preferred that, the DMEM nutrient solutions are high glycoform.
It is preferred that, the application of above-mentioned nutrient solution in vitro in terms of differentiation of stem cells.
As can be seen from the above technical solutions, the embodiment of the present invention has advantages below：
A kind of nutrient solution that the present invention is provided, the nutrient solution that provides of the present invention comprising human chondrocytes culture supernatant and
DMEM nutrient solutions, wherein human chondrocytes culture supernatant contain a variety of rush hUC-MSCs to the thin of cartilage cell's specific differentiation
Intracellular cytokine and active ingredient.The nutrient solution that the present invention is provided can effectively facilitate specificity point of the hUC-MSCs to cartilage cell
Change.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, without having to pay creative labor, may be used also
To obtain other accompanying drawings according to these accompanying drawings.
Fig. 1 shows cellular morphology figure (hUC-MSCs (100 ×) under light microscopic)
Fig. 2 shows hUC-MSCs surface marker flow cytometer detection result schematic diagrams
Fig. 3 shows safranin O-fast green dyeing and II Collagen Type VI ImmunohistochemistryResults Results schematic diagrames
Fig. 4 shows II Collagen Type VI (Col-2) expression comparison schematic diagram
Fig. 5 shows glycosaminoglycan (GAG) expression comparison schematic diagram
Fig. 6 shows SOX-9 expression comparison schematic diagrams
Embodiment
To enable goal of the invention, feature, the advantage of the present invention more obvious and understandable, below in conjunction with the present invention
Accompanying drawing in embodiment, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that disclosed below
Embodiment be only a part of embodiment of the invention, and not all embodiment.Based on the embodiment in the present invention, this area
All other embodiment that those of ordinary skill is obtained under the premise of creative work is not made, belongs to protection of the present invention
Scope.
The invention provides a kind of nutrient solution, human chondrocytes culture supernatant and DMEM nutrient solutions are included.
The invention provides a kind of nutrient solution, human chondrocytes culture supernatant and DMEM nutrient solutions are included.Wherein, it is described
The main function of human chondrocytes culture supernatant in the present invention is to promote specific differentiations of the hUC-MSCs to cartilage cell.
It is preferred that, the human chondrocytes culture supernatant and the volume ratio of DMEM nutrient solutions are：0.5~1.5:1.5~
1.5, preferred volume ratio is 1:1.
The nutrient solution that the present invention is provided, wherein human chondrocytes culture supernatant is preferably prepared by the following：
First, articular cartilage tissue is collected.The articular cartilage tissue is from the articulations digitorum manus for referring to resection operation more.By joint
Cartilaginous tissue is collected in phosphate buffer, and cleaning repeatedly is shredded.The specification of the tissue block shredded be preferably lmm ×
1mm × 1mm or so.Tissue block is transferred in 50mL sterile centrifugation tubes and digested.The process of the digestion digests to add
Liquid carries out digestion 30min, and the digestive juice is preferably 0.05% II Collagenase Type.Cyclic washing, centrifugation after digestion.Then add
Digestive juice carries out water-bath and filtering successively.The water-bath optimum condition is 3-4h of slight oscillatory digestion in 37 DEG C of water-baths.The mistake
The screen cloth of filter is preferably 200 mesh cell screen clothes.Cell suspension is collected into 50mL sterile centrifugation tubes after filtering.Do not digest completely
Cartilaginous tissue proceed digestion.The digestion process is placed in slight oscillatory digestion 1 in 37 DEG C of water-baths to add after digestive juice
~2h, disappears until cartilaginous tissue block, untill solution turned cloudy substantially.The digestive juice is preferably that 0.25% trypsase is molten
Liquid.
Secondly, postdigestive cell is collected, is counted.The quantity of the counting is preferably 1 × 109/L.Will after counting
Cell is inoculated in 10cm cultures, changes liquid 1 time within every 2 days.When primary cell is merged and covers ware bottom more than 80%, digested
And passage.The digestive juice that the digestion process is used is preferably 0.25% trypsase, and the ratio of the passage is preferably 1:
2。
Finally, cells and supernatant to 50mL centrifuge tubes are collected every 48h to be centrifuged.The condition of the centrifugation is preferably
2000rpm/min centrifuges 10min.Aseptically supernatant is taken to be filtered after centrifugation.The filter particle diameter of the filtering is excellent
Elect 0.2~0.24 μm, more preferably 0.22 μm as.Supernatant after filtering is free of cartilage cell through microscopy, the people is made soft
Osteocyte culture supernatant, is saved backup in -20 DEG C of refrigerators.
In the present invention, the cell that act as of the DMEM nutrient solutions provides effective nutritional ingredient.
It is preferred that, the DMEM nutrient solutions include TGF, dexamethasone, L-AA, ITS+Premix
And FBS.It is more highly preferred to, the TGF is any one in β 1 or β 2.It is preferred that, the DMEM nutrient solutions
For high glycoform.
Present invention also offers the application in terms of above-mentioned nutrient solution in vitro differentiation of stem cells.
A kind of nutrient solution that the present invention is provided, the nutrient solution that provides of the present invention comprising human chondrocytes culture supernatant and
DMEM nutrient solutions, wherein human chondrocytes culture supernatant contain a variety of rush hUC-MSCs to the thin of cartilage cell's specific differentiation
Intracellular cytokine and active ingredient.The nutrient solution that the present invention is provided can be effectively increased II Collagen Type VI synthetic quantities, promote hUC-MSCs to
The specific differentiation of cartilage cell.
For the sake of becoming apparent from, it is described in detail below by following examples.
The umbilical cord mesenchymal stem cells culture of embodiment 1
(1) separation of umbilical cord mesenchymal stem cells
1. umbilical cord is derived from healthy puerpera (Serological testing such as hepatitis B, hepatitis, HIV, mycoplasma, syphilis is feminine gender), will
Umbilical cord is placed in the PBS containing 100U/mL penicillin and 100U/mL streptomysins and rinsed 2 times, adds 75% alcohol of precooling, leaching
1-2min is steeped, does not during which stop to stir umbilical cord；
2. addition PBS, which is rinsed 2 times, washes away alcohol.Umbilical cord is cut into 2mm or so segment with tissue shear, every section with eye scissors
Longitudinal direction is opened, and three blood vessels (two arteries, a vein) in umbilical cord are removed with vessel forceps, while removing umbilical cord outer membrane；Will separation
Good umbilical cord is cut into about 1mm with eye scissors3The tissue block of size, takes and is put into right amount in a diameter of 10cm sterile petri dish, cover
The floor space of the culture dish of lid 70%.
3. LONZA human stem cells serum-free medium (Lonza UltraCULTURETM), 5%CO are added2, 37 DEG C, it is full
With the CO that humidity is 95%2Cultivated in incubator.5th~7 day half amount changes culture medium, continues 12~14 days or so full doses of culture and changes
Liquid removes tissue block, while collecting passage culture.
(2) cellular morphology is observed
Cellular morphology is observed under the microscope, as shown in Figure 1.As can be seen from Figure 1 hUC-MSCs grows vigorous, border
It is smooth, nido growth is presented, colony is in polygonal or fusiformis mostly, and the arrangement of colony inner cell is close, and endochylema relatively enriches, nucleus
Greatly, kernel is big.
(3) cell surface antigen is detected
The 3rd generation exponential phase cell is taken, cell separation liquid digests, Flow cytometry surface antigen CD 105,
CD73, CD90, CD34, CD45, CD11b, CD19, HLA-DR expression, Cell-Quest software analysis results.Each sample
Analyze 6000~8000 cells.
The hUC-MSCs cell surface marker expression rates of table 1
| Cell phenotype | CD105 | CD90 | CD73 | HLA-DR | CD34 | CD45 | CD11b | CD19 |
| Positive expression rate (%) | 99.70 | 99.80 | 99.80 | 0.30 | 0.20 | 0.10 | 0.00 | 0.00 |
Such as table 1, shown in Fig. 2, through Flow cytometry, the 3rd generation hUC-MSCs height expresses CD105, CD73 and CD90, low
Expression or the not label such as expression of HLA-DR, CD19, CD11b, CD45 and CD34.As a result hUC-MSCs and other sources are shown
MSCs is essentially identical.
Embodiment 2
(1) the 3rd obtained generation hUC-MSCs is cultivated in Example 1, by 5 × 105Cell/ pipes are inoculated in 15mL centrifuge tubes
Interior, 1000rpm centrifugation 5min make cell is agglomerating to be gathered in centrifugation bottom of the tube, lid unscrews half, are put into 37 DEG C, 5%CO2Training
Support case quiescent culture.
(2) inhaled after 24h and abandon old liquid, change 1mL negative control group culture mediums：DMEM in high glucose nutrient solution containing 10%FBS.Gently
Playing centrifuge tube floats cell mass, and lid unscrews half, is put into 37 DEG C, 5%CO2Incubator quiescent culture.
(3) paraffin section, safranin O-fast green dyeing and II Collagen Type VI SABCs etc. are carried out after culture 21d to cell real
Test.
(4) safranin O-fast green Coloration experiment result is as shown in figure 3, cell section non-coloring.II Collagen Type VIs SABC is real
Test result and show that no II Collagen Type VIs are formed.
Embodiment 3
(1) the 3rd obtained generation hUC-MSCs is cultivated in Example 1, by 5 × 105Cell/ pipes are inoculated in 15mL centrifuge tubes
Interior, 1000rpm centrifugation 5min make cell is agglomerating to be gathered in centrifugation bottom of the tube, lid unscrews half, are put into 37 DEG C, 5%CO2Training
Support case quiescent culture.
(2) inhaled after 24h and abandon old liquid, change 1mL positive controls composition chondrocyte induction liquid：Containing 10 μ g/L transforming growth factor βs
1、10-7Mol/L dexamethasone, 100 μm of ol/L L-AAs, 1%ITS Premix, 10%FBS DMEM in high glucose cultures
Liquid.Flicking centrifuge tube floats cell mass, and lid unscrews half, is put into 37 DEG C, 5%CO2Incubator quiescent culture.
(3) paraffin section, safranin O-fast green dyeing and II Collagen Type VI immune groups are carried out after induction differentiation culture 21d to cell
The experiment such as change.
(4) safranin O-fast green Coloration experiment result is as shown in figure 3, cell section has coloring, but painted areas is smaller, II types
Collagen immunization group experimental result shows II Collagen Type VIs and formed.
Embodiment 4
(1) the 3rd obtained generation hUC-MSCs is cultivated in Example 1, by 5 × 105Cell/ pipes are inoculated in 15mL centrifuge tubes
Interior, 1000rpm centrifugation 5min make cell is agglomerating to be gathered in centrifugation bottom of the tube, lid unscrews half, are put into 37 DEG C, 5%CO2Training
Support case quiescent culture.
(2) inhaled after 24h and abandon old liquid, change 1mL experiment composition chondrocyte induction liquid：Contain 10 μ g/L TGFs by 50%
β1、10-7Mol/L dexamethasone, 100 μm of ol/L L-AAs, 1%ITS Premix, 10%FBS DMEM in high glucose cultures
Liquid, the inductive differentiation medium that 50% human chondrocytes culture supernatant is mixed.Flicking centrifuge tube floats cell mass, lid
Unscrew half, be put into 37 DEG C, 5%CO2Incubator quiescent culture.
(3) paraffin section, safranin O-fast green dyeing and II Collagen Type VI immune groups are carried out after induction differentiation culture 21d to cell
The experiment such as change.
(4) safranin O-fast green Coloration experiment result is as shown in figure 3, cell section has a coloring, and painted areas is significantly greater than pair
Ratio 2, II Collagen Type VI immunohistochemical experiment results show the synthesis of II Collagen Type VIs, and II Collagen Type VI synthetic quantities are significantly more than comparative example
2。
Embodiment 5
Nutrient solution inducing effect is evaluated according to II Collagen Type VI (Col-2), glycosaminoglycan (GAG) and SOX-9 genes
Expression of results is evaluated.Detection process is tested according to the operating method of embodiment 2, embodiment 3 and embodiment 4 to hUC-MSCs
Cultivated or induced into cartilage differentiation, II Collagen Type VI (Col-2), gucosamine are carried out to cell after continuous induction culture 21d
The detection of expression of glycan (GAG) and SOX-9 genes.
Cell total rna extraction is carried out according to TRIzol kit specifications；M-MLV reverse transcription reagent box obtains cDNA.Press
According to SYBR Green quantitative PCR kits specifications to II Collagen Type VI (Col-2), glycosaminoglycan (GAG) and SOX-9 genes
Expression is detected.Reference gene is used as using GAPDH.
The primer used is as shown in table 2.
With 2-△△CTMethod carries out quantitative analysis.
The primer sequence table of table 2
As shown in Figure 4,5, 6, after cell induction culture 21d, embodiment 3, embodiment 4 are (negative with embodiment 2 for experimental result
Control group) compare, into cartilage differentiation cell sign gene II Collagen Type VI (Col-2), glycosaminoglycan (GAG) and SOX-9
Gene expression dose has pole significant difference (* *, P<0.01), embodiment 4 has significant difference (*, p with embodiment 3<
0.05).It can illustrate that the culture liquid energy that the present invention is provided effectively facilitates spies of the hUC-MSCs to cartilage cell according to experimental result
Opposite sex differentiation.
Described above, the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to before
Embodiment is stated the present invention is described in detail, it will be understood by those within the art that：It still can be to preceding
State the technical scheme described in each embodiment to modify, or equivalent substitution is carried out to which part technical characteristic；And these
Modification is replaced, and the essence of appropriate technical solution is departed from the spirit and scope of various embodiments of the present invention technical scheme.
Claims (10)
1. a kind of nutrient solution, it is characterised in that include human chondrocytes culture supernatant and DMEM nutrient solutions.
2. nutrient solution according to claim 1, it is characterised in that the human chondrocytes culture supernatant and DMEM nutrient solutions
Volume ratio be：0.5~1.5:1.5~1.5.
3. nutrient solution according to claim 1, it is characterised in that the human chondrocytes culture supernatant is by the following method
Obtain：
A) articular cartilage is collected, cleaning is sequentially passed through, shreds, centrifuges, filtering, obtain cell suspension；
B) it will be cultivated after cell suspension inoculation, cell fusion is digested, passed to when covering culture dish bottom more than 80%
In generation, obtain supernatant crude product；
C) supernatant crude product is centrifuged, takes supernatant to be filtered, microscopy, obtain the human chondrocytes culture supernatant.
4. nutrient solution according to claim 3, it is characterised in that the screen cloth of the step a) filterings is 180~220 mesh.
5. nutrient solution according to claim 3, it is characterised in that the cell density of the step b) inoculations is 0.5~1.5 ×
109Individual/L.
6. nutrient solution according to claim 3, it is characterised in that the filter particle diameter of the step c) filterings is 0.2~0.24 μ
m。
7. nutrient solution according to claim 1, it is characterised in that the DMEM nutrient solutions comprising TGF, fill in
Meter Song, L-AA, ITS Premix and FBS.
8. nutrient solution according to claim 7, it is characterised in that the TGF is any one in β 1 or β 2
Kind.
9. nutrient solution according to claim 7, it is characterised in that the DMEM nutrient solutions are high glycoform.
10. the application of nutrient solution in vitro in terms of differentiation of stem cells according to claim 1 to 9 any one.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630138A (en) * | 2013-11-06 | 2015-05-20 | 陕西瑞盛生物科技有限公司 | Serum-free cartilage cell culture solution |
| CN105132368A (en) * | 2015-08-17 | 2015-12-09 | 深圳华毓造血干细胞研究有限公司 | Cell inducing culture medium and method for inducing and culturing mesenchyma stem cells |
-
2017
- 2017-05-12 CN CN201710334176.4A patent/CN107099502A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630138A (en) * | 2013-11-06 | 2015-05-20 | 陕西瑞盛生物科技有限公司 | Serum-free cartilage cell culture solution |
| CN105132368A (en) * | 2015-08-17 | 2015-12-09 | 深圳华毓造血干细胞研究有限公司 | Cell inducing culture medium and method for inducing and culturing mesenchyma stem cells |
Non-Patent Citations (2)
| Title |
|---|
| WEI-HONG CHEN等: "In vitro stage-specific chondrogenesis of mesenchymal stem cells committed to chondrocytes", 《ARTHRITIS AND RHEUMATOLOGY》 * |
| 周峰等: "人软骨细胞培养上清诱导脐血间充质干细胞向软骨细胞分化", 《中国组织工程研究》 * |
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