CN109136179A - A kind of co-culture method for improving chondrocyte proliferation activity, maintaining cartilage phenotype - Google Patents
A kind of co-culture method for improving chondrocyte proliferation activity, maintaining cartilage phenotype Download PDFInfo
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- CN109136179A CN109136179A CN201811102683.6A CN201811102683A CN109136179A CN 109136179 A CN109136179 A CN 109136179A CN 201811102683 A CN201811102683 A CN 201811102683A CN 109136179 A CN109136179 A CN 109136179A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1352—Mesenchymal stem cells
- C12N2502/1358—Bone marrow mesenchymal stem cells (BM-MSC)
Abstract
The invention discloses a kind of raising chondrocyte proliferation activity, the co-culture method of maintenance cartilage phenotype.The present invention includes the following steps: step 1, the separation of cartilage cell, culture；Step 2, the separation of mesenchymal stem cell, culture；Step 3, mesenchymal stem cell and cartilage cell are proportionally inoculated into culture utensil and co-culture.Mesenchymal stem cell and cartilage cell co-culture the proliferative capacity that cartilage cell can be improved, and can provide seed cell to be cartilage tissue engineered, provide laboratory data for the development of bioreactor.
Description
Technical field
The present invention relates to a kind of raising chondrocyte proliferation activity, the co-culture method of maintenance cartilage phenotype.
Background technique
Organizational project human disease treatment, rehabilitation, in terms of it is with important application prospects.Organizational project
In the cartilage tissue engineered reparation to articular cartilage defect bring hope.The articular cartilage as caused by wound and various diseases lacks
Damage is clinically very common, and with aging of population, number of patients is growing day by day in China, but the self-regeneration of articular cartilage
Act on it is very limited, therefore the reconstruction of articular cartilage damage be always people thirst for solve problem, articular cartilage reparation
And correlative study is particularly important.
The current repair of cartilage carried out both at home and abroad is rebuild mainly by the method for organizational project.The selection of seed cell is
Most important link and precondition in organizational project.Cartilaginous tissue recovery project using cartilage cell as seed cell, can
Improve the quality of repair tissue.But the cartilage tissue engineered a large amount of seed cell of needs, cartilage cell's quantity of autologous
And limited quality, and to obtain a large amount of cartilage cell the wound of body certainly will be aggravated, the amplification in vitro ability of cartilage cell
It is limited, easily there is " dedifferenting " phenomenon.Therefore cartilage cell is very restricted as cartilage tissue engineered seed cell.
Summary of the invention
The purpose of the present invention is providing regarding to the issue above, a kind of raising chondrocyte proliferation is active, maintains cartilage phenotype
Co-culture method solves tissue engineering bone/cartilage kind effectively to provide excellent seed cell rapidly to be cartilage tissue engineered
The clinic and actual demand problem of daughter cell.
In order to achieve the above objectives, the present invention includes the following steps: step 1, the separation of cartilage cell, culture；Step 2, bone
Separation, the culture of bone marrow-drived mesenchymal stem；Step 3, mesenchymal stem cell and cartilage cell are proportionally inoculated into culture
It is co-cultured in utensil.
The step 3 further include: the cell in culture is loaded, pierces cell by mechanics
Swash.
Mechanical environment in the step 3 are as follows: sine wave, stretch range 5% ~ 15%, 0.5Hz.
Mesenchymal stem cell and the inoculative proportion of cartilage cell are 2:1 in step 3.
The step 1 further include: the identification of cartilage cell.
In the step 1 separation, culture of cartilage cell, the method for identification include: cut mammal joint it is thin-skinned
Cartilage piece is cut into 5mm × 5mm fritter by bone tissue, and PBS liquid cleans 2-3 times；Pancreatin/EDTA that concentration is 0.25% is added,
After 37 DEG C of 30 min of digestion, supernatant is removed, addition concentration is 0.2% II Collagenase Type, after 37 DEG C of digestion 4-6 h, passes through aperture
Impurity is filtered off for 200 mesh screens, 5 min is centrifuged with 1000 r/min, removes supernatant, add the DMEM/F12 containing 15% fetal calf serum
Cell suspension is made in complete medium, is inoculated in culture bottle with the cell concentration of 1 × 109/L, is placed in saturated humidity, 37
DEG C, the CO2 incubator culture of volume fraction 5%, change within every 3 days liquid 1 time, observe cell growth status under inverted phase contrast microscope；To
When cell grows to 80-90%, 0.25% trypsase/EDTA digestion is passed in 1: 2 ratio.The 3rd generation cell is taken, is washed with PBS
2 times, cell climbing sheet is first prepared, Toluidine blue staining is positive, and cartilage cell is prompted to secret out of cartilage matrix, and II Collagen Type VI is immune thin
Born of the same parents' chemical staining is positive.
In the step 2 further include: the identification of mesenchymal stem cell.
The method of separation, culture, the identification of mesenchymal stem cell includes: the bone for taking mammal in the step 2
Cell suspension is made in marrow, and addition has in the centrifuge tube of Percoll separating liquid, and cell suspension and Percoll separating liquid volume ratio are 1
: 1,2000r/min centrifugation 20min draws karyocyte and adds the DMEM/F12 containing 15% fetal calf serum complete after PBS is washed 2 times
Cell suspension is made in culture medium, is inoculated in culture bottle with the cell concentration of 1 × 109/L, is placed in saturated humidity, 37 DEG C, body
The CO2 incubator culture of fraction 5% changes liquid 1 time, observes cell growth status under inverted phase contrast microscope for every 3 days；To cell
When growing to 80-90%, 0.25% trypsase/EDTA digestion is passed in 1: 2 ratio.The 3rd generation cell is taken, is washed 2 times with PBS,
Cell climbing sheet is first prepared, it is fixed, CD31, CD44 immunocytochemical stain are carried out after dehydration, CD31 expression is negative, CD44 table
Up to the positive.
The invention also includes steps 4: the cartilage phenotype detection of chondrocyte proliferation Activity determination, cell.
In the step 4, the method for chondrocyte proliferation Activity determination includes: cartilage cell's vital stain carboxyl fluorescence
Plain acetoacetate dyes, and determines the proliferation of cartilage cell after cell sampling by the fluorescence intensity of flow cytomery cell
Activity, proliferation index=；The method of the cartilage phenotype detection of cell includes: to collect the supernatant of culture cell
Liquid, illustrates to be operated according to glycosaminoglycan (GAG) ELISA immue quantitative detection reagent box, is surveyed under 450nm wavelength with microplate reader
Determine absorbance (OD value), and calculates glycosaminoglycan in sample (GAG) concentration；The cell for collecting culture, using RNAiso kit
The method of offer extracts total serum IgE, then carries out reverse transcription reaction and synthesizes cDNA；Using cDNA as template, using GAPDH as internal reference, press
Illustrate to be operated according to RT-PCR Kit kit, analyzes the expression of 1 gene of Col2 α.
Beneficial effects of the present invention: mesenchymal stem cell and cartilage cell co-culture the increasing that cartilage cell can be improved
Grow ability；The proliferative capacity and cartilage phenotype of cartilage cell is available under mechanical environment culture further increases, and mechanics
Environment culture can promote co-cultured cell to have good cartilage phenotype；Wherein Mechanical loading mode is sine wave, stretch range
5%-10%, 0.5Hz are best；Cell culture processes provided by the invention can provide seed cell to be cartilage tissue engineered, be
The development of bioreactor provides laboratory data.
Detailed description of the invention
A specific embodiment of the invention and its result are further described in detail with reference to the accompanying drawing:
Fig. 1 is the schematic diagram (1: six well culture plate of flexible substrates of Cell viability environment；2: cell；3: flexible substrates film；4:
Negative port)；
Fig. 2 be static culture cartilage cell group and co-cultivation group cartilage cell proliferation index compare statistical chart (n=6, * P <
0.05, * P < 0.001 *)；
Fig. 3 be cartilage cell organize mechanical environment culture after cartilage cell proliferation index compare statistical chart (n=6, * * P <
0.001)；
Fig. 4 compares statistical chart (P < 0.001 n=6, * *) for the proliferation index of cartilage cell after the mechanical environment culture of co-cultivation group；
Fig. 5 compares statistical chart (n=6, * P < 0.05, * * P for the proliferation index of cartilage cell's group and co-cultivation group 4d cartilage cell
< 0.001)；
Fig. 6 compares statistical chart (n=6, * P < 0.05, * * P for the proliferation index of cartilage cell's group and co-cultivation group 6d cartilage cell
< 0.001)；
Fig. 7 is static culture cartilage cell group and co-cultivation group GAG expression statistical chart (P < 0.001 n=6, * *)；
Fig. 8 be static culture cartilage cell group and 1 gene relative expression quantity of co-cultivation group Col2 α compare statistical chart (n=6, * P <
0.05)；
Fig. 9 is mechanical environment cultured cartilage groups of cells GAG expression statistical chart (P < 0.001 n=6, * *)；
Figure 10 is that 1 gene relative expression quantity of mechanical environment cultured cartilage groups of cells Col2 α compares statistical chart (P < 0.05 n=6, *)；
Figure 11 is mechanical environment co-cultivation group cell GAG expression statistical chart (P < 0.001 n=6, * *)；
Figure 12 be 1 gene expression of mechanical environment co-cultivation group cell Col2 α compare statistical chart (n=6, * P < 0.05, * * P <
0.001)；
Figure 13 be cartilage cell's group and co-cultivation group cell GAG expression statistical chart (n=6, * P < 0.05, * * P <
0.001)；
Figure 14 is that statistical chart (P < 0.05 n=6, *) is compared in 1 gene expression of Col2 α of cartilage cell's group and co-cultivation group cell.
Specific embodiment
1, the separation, culture and identification of cartilage cell:
1 monthly age new zealand white rabbit is taken, auricular vein air embolism is lethal, removes limbs skin, and exposure knee joint strikes off joint
The tissue such as muscle, periosteum, synovial membrane that surrounding is adhered to, cuts articular surface cartilaginous tissue, cartilage piece is cut into 5mm × 5mm fritter,
PBS liquid cleans 2-3 times.Concentration is added as 0.25% pancreatin/EDTA, after 37 DEG C of 30 min of digestion, removes supernatant, is added dense
Spending is 0.2% II Collagenase Type, is that 200 mesh screens filter off impurity by aperture, with 1000 r/ after 37 DEG C of digestion 4-6 h
Min is centrifuged 5 min, removes supernatant, adds the DMEM/F12 complete medium containing 15% fetal calf serum that cell suspension is made, with 1 × 109
The cell concentration of a/L is inoculated in culture bottle, is placed in saturated humidity, 37 DEG C, the CO2 incubator culture of volume fraction 5%, and every 3
It is changed liquid 1 time, observes cell growth status under inverted phase contrast microscope.When cell grows to 80-90%, 0.25% trypsase/
EDTA digestion is passed in 1: 2 ratio.The 3rd generation cell is taken, is washed 2 times with PBS, first prepares cell climbing sheet, Toluidine blue staining
The positive, prompts cartilage cell to secret out of cartilage matrix, and II Collagen Type VI immunocytochemical stain is positive.
2, the separation, culture and identification of mesenchymal stem cell
The Limb bone of same new zealand white rabbit, exposure ossis, syringe needle puncture epiphysis and go out bone with DMEM/F12 culture solution
Cell suspension is made in marrow in pulp cavity, and addition has in the centrifuge tube of Percoll separating liquid, cell suspension and Percoll separating liquid
Volume ratio is 1: 1,2000r/min centrifugation 20min, draws karyocyte and adds after PBS is washed 2 times containing 15% fetal calf serum
Cell suspension is made in DMEM/F12 complete medium, is inoculated in culture bottle with the cell concentration of 1 × 109/L, is placed in saturation
Humidity, 37 DEG C, the CO2 incubator culture of volume fraction 5%, are changed liquid 1 time for every 3 days, and cell growth is observed under inverted phase contrast microscope
Situation.When cell grows to 80-90%, 0.25% trypsase/EDTA digestion is passed in 1: 2 ratio.The 3rd generation cell is taken, is used
PBS is washed 2 times, first prepares cell climbing sheet, fixed, and CD31, CD44 immunocytochemical stain, CD31 expression are carried out after dehydration
Feminine gender, CD44 expression are positive.
3, the mechanical environment culture scheme of cell
Cartilage cell's group: cartilage cell is individually inoculated on six well culture plate of flexible substrates；K co-cultivation group: medulla mesenchyma is dry
Cell and cartilage cell are inoculated on six well culture plate of flexible substrates with the co-cultivation of 2:1 ratio, cell inoculation for 24 hours after, pass through
FX-4000 flexible substrates loading system loads the cell in growth, and carrying out mechanical environment culture makes cell by difference
Mechanical stimulation (sine wave, stretch range 5%, 7.5%, 10%, 12.5%, 15%, 0.5Hz), which makes flexibility
Basilar memebrane deformation so that making to be attached to the cell grown on film also occurs corresponding deformation, and then makes cell by corresponding mechanics
Stimulation.Static control experiment is done simultaneously.
Referring to Fig.1, six well culture plate 1 of flexible substrates has cavity to the mechanical environment of cell growth, and cell 2 is in flexible substrates
It is cultivated in the cavity of six well culture plates 1, culture solution is loaded in cavity, the bottom of six well culture plate 1 of flexible substrates is flexible base
Counterdie 3 can be such that flexible substrates film 3 deforms by applying negative pressure at negative port 4, to make to be attached to raw on flexible substrates film 3
Long cell 2 deforms, and makes cell 2 by mechanical stimulation.
4, the cartilage phenotype detection of chondrocyte proliferation Activity determination, cell
4.1, chondrocyte proliferation Activity determination
Cartilage cell is with vital stain Fluoresceincarboxylic acid acetoacetate (carboxyfluorescein diacetate
Succinimidyl ester, CFSE) it dyes, by the fluorescence intensity of flow cytomery cell come really after cell sampling
Determine the proliferation activity of cartilage cell.CFSE is the dyestuff of a kind of pair of cytotoxic, and chemical property is stablized.CFSE once enters thin
It cannot be released from cell after born of the same parents, degradation will not be metabolized.The unique channel of CFSE content reduction is to pass through cell in cell
Multiple fission, CFSE contained in cell enter progeny cell with the proliferation of cell, pass through the flat of flow cytomery cell
Equal fluorescence intensity, fluorescence intensity reduction is more, and proliferation is faster, determines that the proliferation of cartilage cell is living by calculating proliferation index
Property:
Proliferation index= 。
4.1.1 the proliferation activity for improving cartilage cell is co-cultured
According to the average fluorescent strength of flow cytomery cartilage cell, proliferation index is calculated, the results are shown in Table 1.Through uniting
Meter analysis, the proliferation index of co-cultivation group cartilage cell is higher than cartilage cell's group (P < 0.05) when 4d, co-cultivation group cartilage when 6d
The proliferation index of cell is significantly higher than cartilage cell's group (P < 0.001), as shown in Figure 2.Experimental result illustrates that medulla mesenchyma is dry
Cell and cartilage cell co-culture the proliferation activity that cartilage cell can be improved with 2:1 ratio.
The proliferation index (n=6) of 1 cartilage cell of table
| Cartilage cell's group | Co-cultivation group | |
| 4d | 1.69±0.14 | 2.28±0.46 |
| 6d | 3.37±0.59 | 5.16±0.53 |
4.1.2 the proliferation activity of mechanical environment culture and improvement cartilage cell
It is soft according to flow cytomery after cartilage cell's group passes through the mechanical environment culture of FX-4000 flexible substrates loading system
The fluorescence intensity of osteocyte calculates proliferation index, and the results are shown in Table 2.Statistical analysis, when stretch range 5%, 7.5% and 10%
The proliferation index of cartilage cell be apparently higher than static control group (P<0.001), and cartilage is thin in stretch range 12.5% and 15%
When the proliferation index of born of the same parents is significantly less than stretch range 5%, 7.5% and 10% cartilage cell proliferation index (P<0.001), such as Fig. 3 institute
Show.Experimental result illustrates that mechanical stimulation can significantly improve the proliferation activity of cartilage cell in stretch range 5%-10%.
Table 2: cartilage cell organizes the proliferation index (n=6) of cartilage cell after Mechanical loading
| It is static | 5% stretches | 7.5% stretches | 10% stretches | 12.5% stretches | 15% stretches | |
| 4d | 1.69±0.14 | 2.76±0.47 | 2.71±0.44 | 2.74±0.44 | 1.74±0.19 | 1.71±0.15 |
| 6d | 3.37±0.59 | 7.02±0.95 | 7.10±1.13 | 6.98±1.04 | 4.52±0.69 | 4.53±0.68 |
4.1.3 cartilage cell and mesenchymal stem cell mechanical environment co-culture the proliferation activity for improving cartilage cell
After co-cultivation group passes through the mechanical environment culture of FX-4000 flexible substrates loading system, according to flow cytomery cartilage
The fluorescence intensity of cell calculates proliferation index, and the results are shown in Table 3.Statistical analysis, stretch range 5%, 7.5% and 10% mechanics
The proliferation index of cartilage cell is apparently higher than static control group (P < 0.001) after environment co-cultures, and in 12.5% He of stretch range
When the proliferation index of cartilage cell is significantly less than stretch range 5%, 7.5% and 10% when 15% cartilage cell proliferation index (P <
0.001), as shown in Figure 4.Experimental result illustrates that mechanical stimulation can significantly improve co-cultivation cartilage in stretch range 5%-10%
The proliferation activity of cell.
Table 3: the proliferation index (n=6) of mechanical environment co-cultivation group cartilage cell
| It is static | 5% stretches | 7.5% stretches | 10% stretches | 12.5% stretches | 15% stretches | |
| 4d | 2.28±0.46 | 7.26±0.94 | 7.21±0.96 | 7.15±0.96 | 2.71±0.38 | 2.64±0.41 |
| 6d | 5.16±0.53 | 18.86±2.41 | 18.38±1.90 | 18.41±2.03 | 8.93±1.26 | 8.65±1.55 |
4.1.4 cartilage cell's group is compared with co-cultivation group cell-proliferation activity
Cartilage cell's group is for statistical analysis with the proliferation index of co-cultivation group cartilage cell, and cartilage cell and medulla mesenchyma are dry
Cell co-cultures, and the proliferation activity of cartilage cell can be improved, the proliferation index of mechanical environment co-cultivation group cartilage cell is obvious
Higher than mechanical environment cartilage cell group (P < 0.001), as shown in Figure 5, Figure 6.
The experimental results showed that can be improved cartilage thin for mechanical environment culture (sine wave, stretch range 5%-10%, 0.5Hz)
The proliferative capacity of born of the same parents, and mesenchymal stem cell and cartilage cell carry out mechanical environment co-cultivation, Ke Yi great with 2:1 ratio
The proliferative capacity of width raising cartilage cell.
The cartilage phenotype of 4.2 cells detects
Cartilage cell's group: cartilage cell is individually inoculated on six well culture plate of flexible substrates；K co-cultivation group: medulla mesenchyma is dry
Cell and cartilage cell are inoculated on six well culture plate of flexible substrates (as shown in Figure 1) with the co-cultivation of 2:1 ratio, cell inoculation
After for 24 hours, the cell in growth is loaded by FX-4000 flexible substrates loading system, carrying out mechanical environment culture makes carefully
Born of the same parents' stimulation subject to various forces (sine wave, stretch range 5%, 7.5%, 10%, 12.5%, 15%, 0.5Hz), the system is with very
Idling pressure deforms flexible substrates film, so that making to be attached to the cell grown on film also occurs corresponding deformation, and then make cell by
To corresponding mechanical stimulation.In 6d sample detection.Static control experiment is done simultaneously.J collects the supernatant of culture cell, according to sugar
Amine glycan (GAG) ELISA immue quantitative detection reagent box illustrates to be operated, and measures absorbance under 450nm wavelength with microplate reader
(OD value), and calculate glycosaminoglycan in sample (GAG) concentration.K collects the cell of culture, the side provided using RNAiso kit
Method extracts total serum IgE, then carries out reverse transcription reaction and synthesizes cDNA.Using cDNA as template, using GAPDH as internal reference, according to RT-PCR
Kit kit illustrates to be operated, and analyzes the expression of 1 gene of Col2 α.Primer sequence:
GAPDH-F:5 '-TCACCATCTTCCCAGGAGCGA-3 '
GAPDH-R:5 '-CACAATGCCGAAGTGGTCGGT-3 '
Col2 α 1-F:5 '-GTGCGACGACATAATCTGTGAAG-3 '
Col2 α 1-R:5 '-TCCTTTCTGCCCCTTTGGT-3 '
4.2.1 the cartilage phenotype of cell is cultivated
The content of GAG is as shown in table 4 in cell supernatant, and through statistical analysis, when static culture, cartilage cell organizes the table of GAG
It is as shown in Figure 7 up to co-cultivation group (P < 0.001) is apparently higher than.
1 gene expression amount of Col2 α is as shown in table 5 in cell, through statistical analysis, when static culture, and cartilage cell's group
It is as shown in Figure 8 that the expression of 1 gene of Col2 α is higher than co-cultivation group (P < 0.05).
The result shows that the cartilage cell of in vitro culture can synthesize GAG and collagen I I, and co-cultivation group have it is a large amount of undifferentiated
Mesenchymal stem cell, cartilage cell's quantity is few, therefore cell expression GAG and collagen I I is relatively smaller.
Table 4: the expression quantity (n=6) of static culture cartilage cell group and co-cultivation group GAG
| Cartilage cell's group | Co-cultivation group | |
| GAG concentration (ng/L) | 190.50±14.27 | 137.83±11.99 |
Table 5: 1 gene relative expression quantity (n=6) of static culture cartilage cell group and co-cultivation group Col2 α
| Cartilage cell's group | Co-cultivation group | |
| 1 gene relative expression quantity of Col2 α | 13.85±2.16 | 8.98±1.99 |
4.2.2 the cartilage phenotype of mechanical environment culture and improvement cartilage cell
4.2.2.1 the expression of mechanical environment cultured cartilage groups of cells glycosaminoglycan (GAG)
Cartilage cell's group collects cell supernatant detection by 6d after the mechanical environment culture of FX-4000 flexible substrates loading system
Glycosaminoglycan (GAG) concentration, the results are shown in Table 6.Statistical analysis, the sugar of cartilage cell when stretch range 5%, 7.5% and 10%
Amine glycan (GAG) expression is apparently higher than static control group (P < 0.001), and the cartilage cell in stretch range 12.5% and 15%
Glycosaminoglycan (GAG) expression is significantly less than stretch range 5%, 7.5% and 10%(P < 0.001), as shown in Figure 9.Experimental result explanation
Mechanical stimulation can significantly improve the expression of cartilage cell's glycosaminoglycan (GAG) in stretch range 5%-10%.
Table 6: mechanical environment cultured cartilage groups of cells glycosaminoglycan (GAG) expresses (n=6)
| It is static | 5% stretches | 7.5% stretches | 10% stretches | 12.5% stretches | 15% stretches | |
| GAG concentration (ng/L) | 190.50±14.27 | 276.83±19.48 | 277.33±21.64 | 275.67±22.92 | 219.50±17.13 | 219.19±17.43 |
4.2.2.2 the expression of 1 gene of mechanical environment cultured cartilage groups of cells Col2 α
Cartilage cell's group collects cell detection Col2 α 1 by 6d after the mechanical environment culture of FX-4000 flexible substrates loading system
The expression of gene, the results are shown in Table 7.Statistical analysis, 1 gene of cartilage cell Col2 α when stretch range 5%, 7.5% and 10%
Expression be higher than static control group (P < 0.05), and in stretch range 12.5% and 15% 1 gene of Col2 α of cartilage cell table
Up to less than stretch range 5%, 7.5% and 10%(P < 0.05), as shown in Figure 10.Experimental result illustrates mechanical stimulation in stretch range
It can be improved the expression of 1 gene of cartilage cell Col2 α when 5%-10%.
Table 7: 1 gene relative expression quantity (n=6) of mechanical environment cultured cartilage groups of cells Col2 α
| It is static | 5% stretches | 7.5% stretches | 10% stretches | 12.5% stretches | 15% stretches | |
| 1 gene relative expression quantity of Col2 α | 13.85±2.16 | 17.63±2.44 | 16.97±1.76 | 17.03±2.38 | 12.38±1.43 | 11.75±1.90 |
4.2.3 the cartilage phenotype of mechanical environment culture and improvement co-cultivation group cell
4.2.3.1 glycosaminoglycan (GAG) expression of mechanical environment culture co-cultivation group cell
Co-cultivation group cell passes through 6d after the mechanical environment culture of FX-4000 flexible substrates loading system, collects cell supernatant inspection
Glycosaminoglycan (GAG) concentration is surveyed, the results are shown in Table 8.Statistical analysis, cartilage cell when stretch range 5%, 7.5% and 10%
Glycosaminoglycan (GAG) expression is apparently higher than static control group (P < 0.001), and the cartilage cell in stretch range 12.5% and 15%
Glycosaminoglycan (GAG) expression be significantly less than stretch range 5%, 7.5% and 10%(P < 0.001), as shown in figure 11.Experimental result
Illustrate that mechanical stimulation can significantly improve the expression of co-cultivation group cell glycosaminoglycan (GAG) in stretch range 5%-10%.
Table 8: the glycosaminoglycan (GAG) of mechanical environment co-cultivation group cell expresses (n=6)
| It is static | 5% stretches | 7.5% stretches | 10% stretches | 12.5% stretches | 15% stretches | |
| GAG concentration (ng/L) | 137.83±11.99 | 255.33±20.17 | 256.16±22.12 | 254.33±24.65 | 189.67±18.59 | 190.17±18.52 |
4.2.3.2 the expression of 1 gene of mechanical environment co-cultivation group cell Col2 α
Co-cultivation group cell passes through 6d after the mechanical environment culture of FX-4000 flexible substrates loading system, collects cell detection Col2
The expression of 1 gene of α, the results are shown in Table 9.Statistical analysis, 1 base of cartilage cell Col2 α when stretch range 5%, 7.5% and 10%
The expression of cause is apparently higher than static control group (P < 0.001), 1 gene of Col2 α of cartilage cell when stretch range 12.5% and 15%
Expression be less than stretch range 5%, 7.5% and 10%(P < 0.05), as shown in figure 12.Experimental result illustrates that mechanical stimulation is stretching
The expression of 1 gene of co-cultivation group cell Col2 α can be significantly improved when amplitude 5%-10%.
Table 9: the 1 gene relative expression quantity (n=6) of Col2 α of mechanical environment co-cultivation group cell
| It is static | 5% stretches | 7.5% stretches | 10% stretches | 12.5% stretches | 15% stretches | |
| 1 gene relative expression quantity of Col2 α | 8.98±1.99 | 16.98±3.07 | 17.12±2.53 | 17.13±2.98 | 11.38±1.05 | 11.28±1.36 |
4.2.4 cartilage cell's group is compared with co-cultivation group cellular cartilage phenotype
4.2.4.1 cartilage cell's group and glycosaminoglycan (GAG) expression of co-cultivation group cell are for statistical analysis, such as Figure 13 institute
Show, the glycosaminoglycan (GAG) of 5%, 7.5% and 10% stretch range mechanical environment cartilage cell group and co-cultivation group cell expresses nothing
Notable difference (P > 0.05), and glycosaminoglycan (GAG) expression of static co-cultivation group cell is significantly less than static cartilage cell's group
(P < 0.001), 12.5% and 15% stretch co-cultivation group cell glycosaminoglycan (GAG) expression be less than cartilage cell's group (P <
0.05).Illustrate that glycosaminoglycan (GAG) expression of co-cultured cell when stretch range 5%-10% improves, and it is thin to reach cartilage
Born of the same parents are horizontal.
4.2.4.2 cartilage cell's group is for statistical analysis with 1 gene expression of Col2 α of co-cultivation group cell, such as Figure 14 institute
Show, 1 gene of Col2 α of 5%, 7.5%, 10%, 12.5% and 15% stretch range mechanical environment cartilage cell group and co-cultivation group cell
It expresses no significant difference (P > 0.05), and 1 gene expression of Col2 α of static co-cultivation group cell is significantly less than static cartilage cell
Group (P < 0.05).Illustrate that 1 gene expression dose of Col2 α of co-cultured cell when stretch range 5%-15% improves, and reaches cartilage
Cellular level.
The experimental results showed that the cartilage phenotype of co-cultured cell reaches cartilage cell's level, explanation when stretch range 5%-10%
Mechanical environment co-cultivation promotes mesenchymal stem cell and cartilage phenotype occurs, to Chondrocyte Differentiation.Stretch range 5%-
The proliferation activity of co-cultured cell is apparently higher than cartilage cell's group when 10%, thus mesenchymal stem cell and cartilage cell with
2:1 ratio carries out mechanical environment co-cultivation, can greatly improve the proliferation activity of cell, and have good cartilage phenotype.
By above-mentioned experiment it may be concluded that 1. cartilage cells carry out mechanical environment culture, Mechanical loading mode is sine
The proliferative capacity of cartilage cell can be improved in wave, stretch range 5%-10%, 0.5Hz, and it is good that cartilage cell can be maintained to have
Cartilage phenotype.2. mesenchymal stem cell and cartilage cell are inoculated into six well culture plate of flexible substrates with 2:1 ratio, into
Row mechanical environment co-cultures, and Mechanical loading mode is sine wave, and stretch range 5%-10%, 0.5Hz can be improved cartilage cell's
Proliferative capacity, and co-cultured cell has good cartilage phenotype.The cell culture processes that experiment provides can be cartilaginous tissue
Engineering provides seed cell, provides laboratory data for the development of bioreactor.
Claims (10)
1. a kind of co-culture method for improving chondrocyte proliferation activity, maintaining cartilage phenotype, it is characterised in that it includes following
Step:
Step 1, the separation of cartilage cell, culture；
Step 2, the separation of mesenchymal stem cell, culture；
Step 3, mesenchymal stem cell and cartilage cell are proportionally inoculated into culture utensil and co-culture.
2. the co-culture method according to claim 1 for improving chondrocyte proliferation activity, maintaining cartilage phenotype, feature
It is the step 3 further include: load to the cell in culture, make cell by mechanical stimulation under mechanical environment.
3. the co-culture method according to claim 2 for improving chondrocyte proliferation activity, maintaining cartilage phenotype, feature
The mechanical environment being in the step 3 are as follows:
Sine wave, stretch range 5% ~ 15%, 0.5Hz.
4. the co-culture method according to claim 1 for improving chondrocyte proliferation activity, maintaining cartilage phenotype, feature
It is:
Mesenchymal stem cell and the inoculative proportion of cartilage cell are 2:1 in step 3.
5. the co-culture method according to claim 1 for improving chondrocyte proliferation activity, maintaining cartilage phenotype, feature
It is the step 1 further include:
The identification of cartilage cell.
6. the co-culture method according to claim 5 for improving chondrocyte proliferation activity, maintaining cartilage phenotype, feature
It is that the separation, culture of cartilage cell in the step 1, the method for identification include:
Cartilage piece is cut into 5mm × 5mm fritter by the articular surface cartilaginous tissue for cutting mammal, and PBS liquid cleans 2-3 times；
Concentration is added as 0.25% pancreatin/EDTA, after 37 DEG C of 30 min of digestion, removes supernatant, addition concentration is 0.2% II Collagen Type VI
Enzyme after 37 DEG C of digestion 4-6 h, is that 200 mesh screens filter off impurity by aperture, is centrifuged 5 min with 1000 r/min, goes
Clearly, add the DMEM/F12 complete medium containing 15% fetal calf serum that cell suspension is made, be inoculated with the cell concentration of 1 × 109/L
In in culture bottle, being placed in saturated humidity, 37 DEG C, the CO2 incubator culture of volume fraction 5%, liquid 1 time is changed within every 3 days, inverted phase contrast
Microscopically observation cell growth status；When cell grows to 80-90%, 0.25% trypsase/EDTA digestion, by 1: 2 ratio
Example passage；The 3rd generation cell is taken, is washed 2 times with PBS, cell climbing sheet is first prepared, Toluidine blue staining is positive, prompts cartilage cell
Cartilage matrix is secreted out of, II Collagen Type VI immunocytochemical stain is positive.
7. the co-culture method according to claim 1 for improving chondrocyte proliferation activity, maintaining cartilage phenotype, feature
It is in the step 2 further include:
The identification of mesenchymal stem cell.
8. the co-culture method according to claim 7 for improving chondrocyte proliferation activity, maintaining cartilage phenotype, feature
The method for being separation, culture, the identification of mesenchymal stem cell in the step 2 includes:
Take the marrow of mammal to be made cell suspension, addition has in the centrifuge tube of Percoll separating liquid, cell suspension with
Percoll separating liquid volume ratio is that 1: 1,2000r/min is centrifuged 20min, draws karyocyte and adds after PBS is washed 2 times containing 15%
Cell suspension is made in the DMEM/F12 complete medium of fetal calf serum, is inoculated in culture bottle with the cell concentration of 1 × 109/L
It is interior, it is placed in saturated humidity, 37 DEG C, the CO2 incubator culture of volume fraction 5%, is changed within every 3 days liquid 1 time, under inverted phase contrast microscope
Observe cell growth status；When cell grows to 80-90%, 0.25% trypsase/EDTA digestion is passed in 1: 2 ratio；It takes
3rd generation cell is washed 2 times with PBS, first prepares cell climbing sheet, fixed, and CD31, CD44 immunocytochemistry dye are carried out after dehydration
Color, CD31 expression is negative, and CD44 expression is positive.
9. the co-culture method according to claim 1 for improving chondrocyte proliferation activity, maintaining cartilage phenotype, feature
It is that it further includes step 4:
The cartilage phenotype detection of chondrocyte proliferation Activity determination, cell.
10. the co-culture method according to claim 9 for improving chondrocyte proliferation activity, maintaining cartilage phenotype, special
It levies and is in the step 4,
The method of chondrocyte proliferation Activity determination includes: that cartilage cell is dyed with vital stain Fluoresceincarboxylic acid acetoacetate,
Determine the proliferation activity of cartilage cell after cell sampling by the fluorescence intensity of flow cytomery cell, proliferation index=；
The method of the cartilage phenotype detection of cell includes: to collect the supernatant of culture cell, according to glycosaminoglycan (GAG) ELISA
Immue quantitative detection reagent box illustrates to be operated, and is measured under 450nm wavelength with microplate reader absorbance (OD value), and calculate in sample
Glycosaminoglycan (GAG) concentration；The cell for collecting culture extracts total serum IgE using the method that RNAiso kit provides, then carries out
Reverse transcription reaction synthesizes cDNA；Using cDNA as template, using GAPDH as internal reference, illustrate to be grasped according to RT-PCR Kit kit
Make, analyzes the expression of 1 gene of Col2 α.
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