CN109749992A - A kind of mesenchymal stem cell serum-free cultural method - Google Patents
A kind of mesenchymal stem cell serum-free cultural method Download PDFInfo
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Abstract
The invention discloses a kind of mesenchymal stem cell serum-free large-scale cultivation methods.The cultivating system of serum is used compared to the prior art, the present invention uses serum free medium and serum substitute, by spinner culture technique come large-scale culture mescenchymal stem cell, to which entire cultivating system does not introduce animal source component, roller bottle technique also saves man power and material, so that a large amount of mescenchymal stem cells can not only be amplified quickly, and growth of mesenchymal stem cells is in good condition during entire secondary culture of the invention, culture process is stablized, it expands obtained mescenchymal stem cell and maintains higher motility rate, with good potential applicability in clinical practice.
Description
Technical field
The present invention relates to technical field of bioengineering, and in particular to a kind of mesenchymal stem cell serum-free large-scale culture side
Method.
Background technique
Mescenchymal stem cell (Mesenchymal Stem Cells, MSC) be it is a kind of from mesoderm growing early stage mesoderm and
Ectodermic multipotential stem cell, the ability with self-renewing and Multidirectional Differentiation.Mescenchymal stem cell clinically can be applied to
Solve a variety of diseases in the blood system, cardiovascular disease, cirrhosis, the nervous system disease, the excision damage of meniscus of knee joint part
Reparation, autoimmune disease etc. additionally have development prospect in terms of nervous system.
Mescenchymal stem cell most early in being found in marrow, be subsequently found be present in human body occur, many kinds of growth course
In tissue.With the most use at present is the mescenchymal stem cell of derived from bone marrow, and but there are the following problems: with the aging at age,
Mesenchymal stem cell number significantly reduces, Proliferation, Differentiation ability significantly fails；Its preparation process is not easy Quality Control；Transplanting
It may cause immune response when to allosome；Patient is had damage when materials, patient has and cannot acquire when bone marrow disease, even strong
Health donor cannot also extract too many marrow.
Studies have shown that the mescenchymal stem cell in umbilical cord source expresses the peculiar molecular marker of a variety of embryonic stem cells, and
Characteristic big with differentiation potential, proliferative capacity is strong, immunogenicity is low；In addition its materials is convenient, the limit of amoral ethics problem
System, it is easier to preparation of industrialization.Therefore, the ideal that umbilical cord mesenchymal stem cells can not only become mesenchymal stem cell is replaced
For object, and there is bigger application potential.
Therefore efficient amplification is carried out by the large-scale culture of mescenchymal stem cell, to be extensive clinical and scientific research
It is the application project currently to attract attention using sufficient mescenchymal stem cell cell quantity deposit is provided.Currently, most common
Mescenchymal stem cell cultivating system is that DMEM/F12 culture medium is used to carry out body plus volume ratio for 10% fetal calf serum (FBS)
Outer culture and amplification.Although FBS can provide necessary nutriment for mescenchymal stem cell, FBS belongs to animal derived substance,
Comparison of ingredients is complicated, and there are potential risks in clinical application, also increase the probability of cell contamination.
In conclusion the method that people urgently obtain the more mescenchymal stem cells with greater activity of effectively amplification.
Summary of the invention
The shortcomings that method in order to overcome above-mentioned amplification of mesenchymal stem cells of the existing technology, the purpose of the present invention exists
In providing a kind of efficient mesenchymal stem cell serum-free large-scale cultivation method comprising following steps:
1) mescenchymal stem cell (amplification cultivation uses the primary mescenchymal stem cell of preparation, i.e. P0 generation for the first time) measurement is thin
After the sum and motility rate of born of the same parents, the cell inoculation is drawn into cell culture container, inoculum density 5x103-6x103A/cm2, and
The complete medium being made of basal medium and serum substitute is added, wherein containing 3-5% body in the complete medium
The cell culture container, is then put into cell culture rotating device, and adjust revolving speed by the serum substitute of product ratio；
2) when culture on the cell culture rotating device to cell confluency degree reaches 80%-90%, the cell is outwelled
Then the abundant vitellophag of protease is added using brine in culture medium in culture vessel；
3) it is fully retracted, after cell rounding after cell pseudopodium, the complete medium is added to terminate digestion；Then it blows and beats
It to obtain mesenchyma stem cell suspension, is collected in centrifuge tube and carries out centrifugation, add fresh complete training after abandoning supernatant
It supports base weight and hangs cell precipitation, (what is obtained by P0 generation amplification is P1 generation, and successively continuous passage is then gradually to a new generation after amplification
Be incremented by P2 generation, P3 generation, etc.) mescenchymal stem cell be measured by sampling cell sum and motility rate；
4) using the mescenchymal stem cell of new generation repetition step 1) after above-mentioned amplification to 3) progress secondary culture, and feelings are regarded
It repeatedly passes on depending on condition to be expanded on a large scale.
In some embodiments of the invention, the mescenchymal stem cell is that the mesenchyma obtained from source of people umbilical cord tissue is done
Cell.
In some embodiments of the invention, the cell culture container is rolling bottle, and cell culture rotating device is CO2
Roller bottle machine draws 1.0 × 10 in step 1)7-1.5×107A cell inoculation is to 1900cm2The cell culture of surface area
In rolling bottle, and complete medium described in 300-500mL is added, then the rolling bottle is put into CO2In roller bottle machine, and adjusts revolving speed and be
10-30rpm。
In the embodiment above of the present invention, the basal medium be α-MEM basal medium such as Corning,
The product of Hyclone or BI board, and the serum substitute is the production of GMP grades of serum substitute such as Helios or PALL board
Product.
In the embodiment above of the present invention, the cell culture container is rolling bottle, and cell culture rotating device is CO2
Roller bottle machine is cultivated 3-5 days on the roller bottle machine in step 2), when cell confluency degree reaches 80%-90%, is outwelled in rolling bottle
Culture medium, using brine 2-3 times, then be added protease digestion cell.
Further, it outwells and turns when culture on the roller bottle machine to cell confluency degree reaches 85%-90% in step 2)
Then TrypLE is added using brine 2 times in culture medium in bottleTMProtease digestion cell 3-5min.
In the embodiment above of the present invention, complete medium described in 20-50mL is added in step 3) to terminate digestion；
Then it blows and beats to obtain mesenchyma stem cell suspension, is collected in 50mL centrifuge tube and is centrifuged 4-8min at 1300-1600rpm,
The fresh complete medium resuspension cell precipitation of 40mL is added after abandoning supernatant.
Further, after the cell described in step 3) is terminated digestion, the mesenchyma being collected in the centrifuge tube is dry
Cell suspension is centrifuged 5min at 1500 rpm.
In the embodiment above of the present invention, depending on the circumstances in step 4), it is extensive to carry out to carry out 6-18 passage
Amplification.
Beneficial effect
The cultivating system of serum, mesenchymal stem cell serum-free large-scale culture of the invention are used compared to the prior art
Method uses serum free medium and serum substitute, whole by spinner culture technique come large-scale culture mescenchymal stem cell
A cultivating system avoids introducing animal source component, and roller bottle technique also saves man power and material, so that without can be quickly
A large amount of mescenchymal stem cells are amplified, and during entire secondary culture of the invention, growth of mesenchymal stem cells state is good
Good, culture process is stablized, and expands obtained mescenchymal stem cell and maintains higher motility rate, before having good clinical application
Scape.
Detailed description of the invention
Fig. 1 is the growth curve chart of each generation mescenchymal stem cell quantity of P1-P8 successively expanded in embodiment 3.
Fig. 2 is the Cell viability figure of each generation mescenchymal stem cell of P1-P8 successively expanded in embodiment 3.
Fig. 3 is the growth curve chart of each generation mescenchymal stem cell quantity of P1-P8 successively expanded in embodiment 4.
Fig. 4 is the Cell viability figure of each generation mescenchymal stem cell of P1-P8 successively expanded in embodiment 4.
Fig. 5 is the growth curve chart of each generation mescenchymal stem cell quantity of P1-P8 successively expanded in embodiment 5.
Fig. 6 is the Cell viability figure of each generation mescenchymal stem cell of P1-P8 successively expanded in embodiment 5.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means.
Embodiment
Embodiment 1: the acquisition of umbilical cord tissue fritter
1) the mature caesarean birth healthy newborn umbilical cord that will be handled well removes the ligation both ends of umbilical cord, and will with eye scissors
The interlude of the long 10-15cm is cut into the umbilical cord tissue section of 2-3cm long, with containing 1% dual anti-(Pen .- Strep mixed solution)
Physiological saline rinse the umbilical cord tissue section repeatedly to remove bloodstain.
2) above-mentioned umbilical cord tissue section is splitted, and removes artery and vein blood vessel (2 arteries and 1 vein) and epidermis group
Knit, obtain umbilical cord China Tong Shi glue tissue segments, after being rinsed repeatedly containing 1% dual anti-physiological saline, shredded into about 3mm ×
The magnificent Tong Shi glue tissue block of 3mm × 1mm.
Embodiment 2: the adhere-wall culture of magnificent Tong Shi glue tissue block and the acquisition of primary mescenchymal stem cell
1) according to 1 piece/cm2The magnificent Tong Shi glue tissue block of embodiment 1 is seeded in diameter 10cm Tissue Culture Dish and is tiled
It is adherent, tissue block described in about 30-50 block is added in each culture dish, appropriate tissue dried by air block is so that it reduces moisture, adherent jail
Gu.
2) 5mL is gently added into each culture dish by basal medium (α-MEM Cornig) and serum substitute
The complete medium (serum substitute wherein containing 3% volume ratio) of (GMP grades of Helios) composition, tissue block not blown
It rises, is subsequently placed into 37 DEG C, 5%CO2Incubator in be incubated for culture；Fresh complete medium 5mL is replaced after culture 5 days, is continued
Stationary culture 5-9 days, the complete medium during which more renewed every 3-4 days；
3) it observes largely after the mescenchymal stem cell climbed out of in magnificent Tong Shi glue tissue block, outwells culture medium and using life
It manages salt to wash 2 times, then uses TrypLETM(the Gibco board product of Thermo Fischer Scient Inc., the U.S.) between climbing out of to filling
Matter stem cell carries out sufficiently digestion about 3-5min, is fully retracted to pseudopodium, cell rounding, is added described in 20-50mL and cultivates completely
Base terminates digestion, then blows and beats to obtain mesenchyma stem cell suspension, draws cell suspension and is added in 50ml centrifuge tube
It is centrifuged 5min under 1500rpm, adds the fresh complete medium resuspension cell precipitation of 40mL after abandoning supernatant；
4) by the postdigestive mescenchymal stem cell of collection using (100-200 μm) of strainer filtering, to remove cell suspension
The magnificent Tong Shi glue tissue block of middle remnants, the filtered cell of gained are the primary mescenchymal stem cell (P0 generation) prepared.
Embodiment 3: the large-scale culture of mescenchymal stem cell
1) the primary mescenchymal stem cell of source of people umbilical cord that embodiment 2 harvests, i.e. in P0 generation, measure cell by cell counter
Sum and Trypan Blue calculate Cell viability after, draw 1.2 × 107A cell inoculation is to 1900cm2Surface area it is thin
Born of the same parents cultivate rolling bottle (clean spy TCB032002,2L, 1900cm2Rolling bottle；For the product of Guangzhou Jie Te biofiltration limited liability company)
In, and be added 400mL be made of basal medium (α-MEM Cornig) and serum substitute (GMP grades of Helios) it is complete
Culture medium (wherein contains 3% serum substitute), then the rolling bottle is put into CO2Roller bottle machine (GPJ-10000, CO2Roller bottle machine；For
The product of spring and autumn Electron equipment Co., Ltd) in, adjustment revolving speed is 20rpm；
2) it is cultivated 3-5 days on roller bottle machine, when cell confluency degree reaches 80%-90%, outwells the culture medium in rolling bottle, make
With brine 2 times, TrypLE is then addedTMAbundant vitellophag 3-5min；
3) it is fully retracted, after cell rounding after cell pseudopodium, complete medium described in 40mL is added to terminate digestion；Then
Piping and druming is collected in 50mL centrifuge tube to obtain mesenchyma stem cell suspension and is centrifuged 5min at 1500 rpm, abandons after supernatant and adds again
Enter the fresh complete medium of 40mL and cell precipitation be resuspended, to after amplification a new generation (what is obtained by P0 generation amplification is P1 generation,
Successively continuous passage be then gradually incremented by P2 generation, P3 generation, etc.) mescenchymal stem cell be measured by sampling cell sum and motility rate；
4) step 1) is repeated to 3) progress secondary culture, Zhi Daochuan using the mescenchymal stem cell of new generation after above-mentioned amplification
In generation, arrives P8 generation.
Table 1: the quantity and motility rate of each generation mescenchymal stem cell of the P1-P8 successively expanded are (in following table, such as in the column P1
P1 refer to the generation of thesocyte, i.e. initial inoculation cell is P0 generation, and thesocyte is the P1 generation after amplification)
Embodiment 4: the large-scale culture of mescenchymal stem cell
1) the primary mescenchymal stem cell of source of people umbilical cord that embodiment 2 harvests, i.e. in P0 generation, measure cell by cell counter
Sum and Trypan Blue calculate Cell viability after, draw 1.5 × 107A cell inoculation is to 1900cm2Surface area it is thin
Born of the same parents cultivate rolling bottle (clean spy TCB032002,2L, 1900cm2Rolling bottle；For the product of Guangzhou Jie Te biofiltration limited liability company)
In, and be added 500mL be made of basal medium (α-MEM Cornig) and serum substitute (GMP grades of Helios) it is complete
Culture medium (serum substitute wherein containing 3% volume ratio), then the rolling bottle is put into CO2Roller bottle machine (GPJ-10000, CO2
Roller bottle machine；For the product of spring and autumn Electron equipment Co., Ltd) in, adjustment revolving speed is 30rpm；
2) it is cultivated 3-5 days on roller bottle machine, when cell confluency degree reaches 80%-90%, outwells the culture medium in rolling bottle, make
With brine 2 times, TrypLE is then addedTMAbundant vitellophag 3-5min；
3) it is fully retracted, after cell rounding after cell pseudopodium, complete medium described in 50mL is added to terminate digestion；Then
Piping and druming is collected in 50mL centrifuge tube to obtain mesenchyma stem cell suspension and is centrifuged 4min at 1600 rpm, abandons after supernatant and adds again
Enter the fresh complete medium of 40mL and cell precipitation be resuspended, to after amplification a new generation (what is obtained by P0 generation amplification is P1 generation,
Successively continuous passage be then gradually incremented by P2 generation, P3 generation, etc.) mescenchymal stem cell be measured by sampling cell sum and motility rate；
4) step 1) is repeated to 3) progress secondary culture, Zhi Daochuan using the mescenchymal stem cell of new generation after above-mentioned amplification
In generation, arrives P8 generation.
Table 2: the quantity and motility rate of each generation mescenchymal stem cell of the P1-P8 successively expanded are (in following table, such as in the column P1
P1 refer to the generation of thesocyte, i.e. initial inoculation cell is P0 generation, and thesocyte is the P1 generation after amplification)
Embodiment 5: the large-scale culture of mescenchymal stem cell
1) the primary mescenchymal stem cell of source of people umbilical cord that embodiment 2 harvests, i.e. in P0 generation, measure cell by cell counter
Sum and Trypan Blue calculate Cell viability after, draw 1.0 × 107A cell inoculation is to 1900cm2Surface area it is thin
Born of the same parents cultivate rolling bottle (clean spy TCB032002,2L, 1900cm2Rolling bottle；For the product of Guangzhou Jie Te biofiltration limited liability company)
In, and be added 300mL be made of basal medium (α-MEM Cornig) and serum substitute (GMP grades of Helios) it is complete
Culture medium (serum substitute wherein containing 3% volume ratio), then the rolling bottle is put into CO2Roller bottle machine (GPJ-10000, CO2
Roller bottle machine；For the product of spring and autumn Electron equipment Co., Ltd) in, adjustment revolving speed is 10rpm；
2) it is cultivated 3-5 days on roller bottle machine, when cell confluency degree reaches 80%-90%, outwells the culture medium in rolling bottle, make
With brine 2 times, TrypLE is then addedTMAbundant vitellophag 3-5min；
3) it is fully retracted, after cell rounding after cell pseudopodium, complete medium described in 20mL is added to terminate digestion；Then
Piping and druming is collected in 50mL centrifuge tube to obtain mesenchyma stem cell suspension and is centrifuged 8min at 1300rpm, abandons after supernatant and adds again
Enter the fresh complete medium of 40mL and cell precipitation be resuspended, to after amplification a new generation (what is obtained by P0 generation amplification is P1 generation,
Successively continuous passage be then gradually incremented by P2 generation, P3 generation, etc.) mescenchymal stem cell be measured by sampling cell sum and motility rate；
4) step 1) is repeated to 3) progress secondary culture, Zhi Daochuan using the mescenchymal stem cell of new generation after above-mentioned amplification
In generation, arrives P8 generation.
Table 3: the quantity and motility rate of each generation mescenchymal stem cell of the P1-P8 successively expanded are (in following table, such as in the column P1
P1 refer to the generation of thesocyte, i.e. initial inoculation cell is P0 generation, and thesocyte is the P1 generation after amplification)
Since the present invention uses serum free medium and serum substitute, by spinner culture technique come between large-scale culture
Mesenchymal stem cells, so that entire cultivating system does not introduce animal source component, roller bottle technique also saves man power and material, so that
Between each generation of P1-P8 that can not only be amplified a large amount of mescenchymal stem cells quickly, and successively expand shown in the table 1-3
The growth of cell shown in the quantity and motility rate and Fig. 1-6 of mesenchymal stem cells figure and Cell viability figure are it is found that in entire passage training
During supporting, growth of mesenchymal stem cells is in good condition, culture process is stable, expands obtained mescenchymal stem cell and maintains
Higher motility rate has good potential applicability in clinical practice.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of mesenchymal stem cell serum-free cultural method, which is characterized in that comprise the following steps:
1) by after the sum and motility rate of mescenchymal stem cell measurement cell, the cell inoculation is drawn into cell culture container,
Inoculum density 5x103-6x103A/cm2, and the complete medium being made of basal medium and serum substitute is added, wherein
Then the cell culture container is put into cell training by the serum substitute containing 3-5% volume ratio in the complete medium
It supports in rotating device, and adjusts revolving speed；
2) when culture on the cell culture rotating device to cell confluency degree reaches 80%-90%, the cell culture is outwelled
Then the abundant vitellophag of protease is added using brine in culture medium in container；
3) it is fully retracted, after cell rounding after cell pseudopodium, the complete medium is added to terminate digestion；Then it blows and beats to obtain
It to mesenchyma stem cell suspension, is collected in centrifuge tube and carries out centrifugation, add fresh complete medium after abandoning supernatant
Cell precipitation is resuspended, the mescenchymal stem cell of new generation after amplification is measured by sampling the sum and motility rate of cell；
4) repeat step 1) to 3) carrying out secondary culture using the mescenchymal stem cell of new generation after above-mentioned amplification, and optionally and
Fixed repeatedly passage to be expanded on a large scale.
2. the method as described in claim 1, which is characterized in that the mescenchymal stem cell is to obtain from source of people umbilical cord tissue
Mescenchymal stem cell.
3. the method as described in claim 1, which is characterized in that the cell culture container is rolling bottle, and cell culture rotates
Device is CO2Roller bottle machine draws 1.0 × 10 in step 1)7-1.5×107A cell inoculation is to 1900cm2Surface area
In cell-culturing rotating bottle, and complete medium described in 300-500mL is added, then the rolling bottle is put into CO2In roller bottle machine, and adjust
Turn over speed is 10-30rpm.
4. method as claimed in claim 3, which is characterized in that the basal medium is α-MEM basal medium, and described
The serum substitute that serum substitute is GMP grades.
5. method as claimed in claim 4, which is characterized in that the α-MEM basal medium be Corning, Hyclone or
The product of BI board, and the serum substitute is the product of Helios or PALL board.
6. method as claimed in claim 3, which is characterized in that cultivated 3-5 days on the roller bottle machine in step 2), when thin
When born of the same parents' convergence degree reaches 80%-90%, the culture medium in rolling bottle is outwelled, using brine 2-3 times, protease is then added
Abundant vitellophag.
7. method as claimed in claim 6, which is characterized in that cultivated on the roller bottle machine to cell confluency in step 2)
When degree reaches 85%-90%, the culture medium in rolling bottle is outwelled, using brine 2 times, TrypLE is then addedTMProtease
Vitellophag 3-5min.
8. method as claimed in claim 6, which is characterized in that be added in the step 3) complete medium described in 20-50mL with
Terminate digestion；Then blow and beat to obtain mesenchyma stem cell suspension, be collected in 50mL centrifuge tube at 1300-1600rpm from
Heart 4-8min adds the fresh complete medium resuspension cell precipitation of 40mL after abandoning supernatant.
9. method according to claim 8, which is characterized in that after the cell described in step 3) is terminated digestion, be collected in
Mesenchyma stem cell suspension in the centrifuge tube is centrifuged 5min at 1500 rpm.
10. method according to claim 8, which is characterized in that depend on the circumstances in step 4) carry out 6-18 times passage with
It is expanded on a large scale.
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| CN112725264A (en) * | 2021-01-21 | 2021-04-30 | 华夏源细胞工程集团股份有限公司 | Method for inducing differentiation of human mesenchymal stem cells into lipid in vitro |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110923203A (en) * | 2019-12-03 | 2020-03-27 | 北京银丰鼎诚生物工程技术有限公司 | Amplification culture process of umbilical cord mesenchymal stem cells |
| CN112725264A (en) * | 2021-01-21 | 2021-04-30 | 华夏源细胞工程集团股份有限公司 | Method for inducing differentiation of human mesenchymal stem cells into lipid in vitro |
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