CN108753710A - A kind of Serum-free complete medium and its application - Google Patents
A kind of Serum-free complete medium and its application Download PDFInfo
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Abstract
The present invention provides a kind of Serum-free complete medium and its application, the Serum-free complete medium, including successively or following two culture mediums for being used in mixed way：Wherein, culture medium I is that addition L-Glutamine, 1%-4% can effectively facilitate cell adherence and the serum substitute of extension, a concentration of 2mM/L of L-Glutamine in basal medium；Medium ii is that the serum substitute of L-Glutamine, 7%-10% for cultivating ESC and iPSC, a concentration of 2mM/L of L-Glutamine are added in basal medium.The present invention also provides the serum-frees to support application of the base in cultivating mescenchymal stem cell completely.Medium component used in the present invention simply prepares simplicity, and the deciduous teeth mescenchymal stem cell cultural method provided is conducive to vitro culture of human deciduous teeth mescenchymal stem cell, maintains " dryness " and Multidirectional Differentiation ability of stem cell.
Description
Technical field
The present invention relates to technical field of cell culture, and in particular to a kind of Serum-free complete medium and its application.
Background technology
Stem cell is generally defined as the Clone formation cell of energy self-renewing and polyphyly differentiation, can be incited somebody to action according to cell origin
Stem cell is divided into embryonic stem cell and adult stem cell.When egg division of being fertilized develops into blastocyst, the cell of inner cell mass is
For embryonic stem cell.Embryonic stem cell has totipotency, with self-renewing and can have the organized ability of institute in vivo that is divided into, but
It is that certain ethics problem is involved to its research and utilization.Many tissues and organ of the adult stem cell from adults,
Under given conditions, adult stem cell can carry out certain program differentiation, form new functioning cell, to make tissue and
Organ keeps the dynamic equilibrium of growth and decline.A variety of stem cells, such as intraepithelial stem cell, tooth are had also discovered in the oral cavity
The stem cell etc. in stem cell and parodontium in marrow, mescenchymal stem cell also related with tooth linked groups.They are not
Only self-renewal capacity, moreover it is possible to which polyphyly is divided into various tissues.Using the autologous stem cells of patient, can be divided into including culture
Various tissues including dentine, dental pulp provide unlimited possibility for the injury repair of Oral and Maxillofacial Surgery.
Peel off deciduous teeth dental pulp stem cell (SHED：Stem cells from exfoliated deciduous teeth) be
The adult mesenchymal stem cells with stronger proliferative capacity and multi-lineage potential separated from mankind's deciduous teeth dental pulp, have
Scholar is also known as immature dental pulp stem cell (immature dental pulp stem cells).SHED is not only expressed
The mescenchymal stem cells mark such as STRO-1, CD29, CD44, CD73, CD90, CD105, MUC18, CD146 and CD166, is also expressed
Embryonic stem cell mark (Oct4 and Nanog), stage specific embryonic antigen (SSEA-3 and SSEA-4) and tumour identification are anti-
Former (TRA-1-60 and TRA-A-81) etc..Compared with dental pulp stem cell, when high deciduous teeth stem cells hyperplasias activity, the population doublings of falling off
Between it is short, clonality is strong, the inflammatory factors expression such as MMP1, TIMP1, MMP2, TIMP2 and IL-6 is higher in cell.
Multidirectional Differentiation ability is similar to DPSC, including skeletonization, at dentine, at fat, at cartilage, at flesh etc., and SHED cells at blood
Pipe is better than DPSC at Neural Differentiation ability.Since it can express a variety of nerve cell marks, induced in Neuronal induction media
Under, the expression of the significant albumen of the nerve cells such as III-tubulin of β, GAD raises, and cell process is more, forms similar god
Ball sample colony through stem cell.In the blood vessels under the stimulation of skin growth factor, fall off deciduous teeth stem cell can VEGF expression 2, CD31,
The significant albumen of the blood vessel endotheliums such as blood vessel endothelium cadherin forms capillary.In addition SHED cells also have induction periphery
Host is differentiated to form the characteristic of bon e formation cell.Internal transplantation experiments can form dentine dental pulp sample tissue, at dentine sample
Cell and osteoid tissue, but complete pulpodentinal complex's spline structure cannot be formed.Prompted above SHED be it is a kind of more
Stem cell that is original, belonging to mesoderm growing early stage.
According to《Stem cell medicine quality control and preclinical study guideline》Requirement：Stem cell medicine preparation is made
Medium component should have enough purity and meet it is sterile, without pathogenic microorganisms and endotoxic quality standard, it is remaining
Culture medium should have no adverse effects to receptor, that is, in the case of the normal growth for meeting stem cell, not influence the biology of stem cell
Active (" dryness " and differentiation capability that include stem cell).In stem cell medicine preparation process, it should avoid using antibiosis as possible
Element；In addition to special circumstances, user source or animal derived serum in stem cell incubation should be avoided as far as possible, must not be used
Allogeneic human serum or blood plasma.Must such as animal blood serum be used it is ensured that it is polluted without particular animals borne virus.
After it experienced natural medium, synthetic media, serum free medium and free serum culture become current cell
One main trend in culture field.The program that purifying and the various cellular products of identification can be simplified using free serum culture, avoids disease
It is endangered caused by poison pollution.Its component of the culture medium of progress free serum culture should meet claimed below：(1) contain basis culture
Base, containing amino acid, vitamin, inorganic salts and glucose etc.；(2) cell adhesion factor：Collagen and fibronectin etc.；(3)
Hormone and growth factor：Promote cell mitogen and amplification in vitro, including insulin, epidermal growth factor etc.；(4) egg is combined
In vain：Such as transferrins or albumin；(5) trace element and antioxidant：Sodium selenite, beta -mercaptoethanol etc..
Ultroser G are a kind of serum substitutes produced by Pall companies, and biological activity is fetal calf serum
(FBS) 5 times.The serum substitute is successfully used for the in vitro culture of attached cell and the dye of amniocyte in pre-natal diagnosis
Colour solid group type analysis.KnockOut Serum Replacement (KnockOut SR) be by GIBCO companies produce it is a kind of at
Point specific, medium additives of no fetal calf serum ingredient can support being incubated on feeder layer from animal or people
Multipotential stem cell proliferation, this serum substitute possesses nearly 20 years usage histories, and this kind of serum substitute is available
In preclinical and clinical research.
Application No. is a kind of 201710241721.5 Chinese patent application " human umbilical cord mesenchymal stem cells free serum cultures
Base " disclose it is a kind of including Ultroser G, bFGF, L-Glutamine Serum-free complete medium composition.Although the Shen
Please in only with Ultroser G, a kind of this serum substitute can cultivate mescenchymal stem cell well, but embodiment only gives
The upgrowth situation for cultivating a generation cell, not to the hereditary shape of stem cell after the culture medium long-term cultivation mescenchymal stem cell
State, activity, differentiation state are explained.
Application No. is 201610877358 Chinese patent application, " a kind of culture medium and its application and culture dental pulp are dry thin
Born of the same parents " disclose a kind of serum free medium of culture dental pulp mescenchymal stem cell, except blood serum substituting beyond the region of objective existence also added 9 kinds additionally
Substance, including cell factor, albumin etc., nutrient media components are complicated.According to《It stem cell medicine quality control and preclinical grinds
Study carefully guideline》Requirement：If the serum composition containing someone in culture medium, such as albumin, transferrins and various cell factors
Deng, it should be understood that its source, lot number, quality arbitration return on qualification, and use as possible national approved can clinical application product.
Invention content
In order to solve the problems existing in the prior art, the present invention provides a kind of Serum-free complete medium and its applications.
In order to achieve the above object, the present invention is achieved by the following technical programs：
A kind of Serum-free complete medium, including successively or following two culture mediums for being used in mixed way：
Wherein, culture medium I is that addition L-Glutamine, 1%-4% can effectively facilitate cell adherence in basal medium
With the serum substitute of extension, a concentration of 2mM/L of L-Glutamine；
Medium ii is that the blood of L-Glutamine, 7%-10% for cultivating ESC and iPSC is added in basal medium
Clear substitute, a concentration of 2mM/L of L-Glutamine.
Preferably, serum substitute is Ultroser G in the culture medium I.
Preferably, serum substitute is KnockOut Serum Replacement in the medium ii.
Preferably, the basal medium is selected from α-MEM, DMEM, DMDM/F12 or IMEM/F12.Culture medium I and culture
Basal medium used in base II can be the same or different.
Preferably, in medium ii can also include EGF, bFGF, a concentration of 10ng/ml of the EGF, the bFGF's
A concentration of 10ng/ml.Basic fibroblast growth factor (bFGF) is a kind of mitogen, the increasing to mescenchymal stem cell
Growing has apparent facilitation, and epidermal growth factor (EGF) can promote derived mesenchymal stem cells in vitro to be proliferated.
The present invention also provides the serum-frees to support application of the base in cultivating mescenchymal stem cell completely, is especially training
Support the application in deciduous teeth mescenchymal stem cell.
The present invention also provides a kind of culture method in serum-free of deciduous teeth mescenchymal stem cell, the method uses such as right
It is required that stem cell of the mixed culture medium culture of the culture medium I and medium ii described in 1 per generation, wherein trains in mixed culture medium
The serum substitute ratio for supporting base I and medium ii is 1:1-1:5.
The culture method in serum-free of another kind deciduous teeth mescenchymal stem cell provided by the invention, the method includes following steps
Suddenly:
A, after being incubated the cell 4h-24h after passage using culture medium I as described in claim 1, this kind culture is removed
Base；
B, continue culture to cell confluency degree using medium ii as described in claim 1 and reach 80%-90%.
C, it passes on every time all with above-mentioned training method.
The culture method in serum-free of another kind deciduous teeth mescenchymal stem cell provided by the invention, the method use such as right
It is required that the proportional region of the mixed culture medium of the culture medium I and medium ii described in 1, culture medium I and medium ii is 1:5-3:
7, and with the increase of culture generation, the ratio of culture medium I is to gradually increase in mixed culture medium.
The beneficial effects of the invention are as follows：
Medium component used in the present invention simply prepares simplicity, and the serum substitute used has permanent research
Usage history, wherein KnockOut SR are also approved for clinic, avoid the pollution of particular animals borne virus.The present invention
The deciduous teeth mescenchymal stem cell cultural method of offer is conducive to vitro culture of human deciduous teeth mescenchymal stem cell, maintains stem cell
" dryness " and Multidirectional Differentiation ability.
Description of the drawings
Fig. 1 is the adherent sight of cell after medium culture deciduous teeth mescenchymal stem cell 4-5 hours with the G containing 2%Ultroser
Examine result；
Fig. 2 is the deciduous teeth mescenchymal stem cell with the medium culture of the Replacement of Serum containing 10%KnockOut
The adherent observation result of cell after cultivating 4-5 hours；
Fig. 3 is with the adherent observation knot of cell after medium culture deciduous teeth mescenchymal stem cell 4-5 hour containing 10%FBS
Fruit；
Fig. 4 is deciduous teeth mescenchymal stem cell morphologic observation result after 3 kinds of different culture media cultures 18 hours；
Fig. 5 is the medium culture deciduous teeth mescenchymal stem cell using the Replacement of Serum containing 10%KnockOut
The cellular morphology picture of different time points；
Fig. 6 be deciduous teeth mescenchymal stem cell with containing 10%FBS 3 generations of medium culture and with containing 2%Ultroser
Cellular morphology picture after 3 generations of medium culture of G；
Fig. 7 is that deciduous teeth mescenchymal stem cell makes the culture medium containing 2% Ultroser G promote cell adherent 6 in embodiment 1
The adherent form picture of cell after hour；
Fig. 8 is that deciduous teeth mescenchymal stem cell uses the cellular morphology figure after 3 generations of training method culture of embodiment 1
Piece；
Fig. 9 is that deciduous teeth mescenchymal stem cell uses cell after the generation 6 hours of training method culture first of embodiment 2
Adherent form picture；
Figure 10 be embodiment 2 cultivate after 3 generations with thin after 3 generations of medium culture containing 10% fetal calf serum
Born of the same parents' form picture；
Figure 11 is that deciduous teeth mescenchymal stem cell uses after 3 generations of training method culture of embodiment 3 and containing fetal calf serum
3 generations of medium culture culture after cellular morphology picture.
Specific implementation mode
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention,
Technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is the present invention one
Divide embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making
The every other embodiment obtained under the premise of creative work, shall fall within the protection scope of the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.Examination as used in the following examples
Agent material etc. is commercially available products unless otherwise specified.
Ultroser G used in the present invention are the solution prepared after dissolving, the specification provided according to Pall companies
Ultroser G freeze-dried powders are completely dissolved in the sterile no heat source waters for injection of 20ml, progress is aseptic subpackaged, is stored in -20 DEG C
In refrigerator, low temperature thaws when use.
Comparative example：3 kinds of culture mediums cultivate deciduous teeth mescenchymal stem cell respectively
3 kinds of culture mediums are used in comparative example：
1. the DMEM culture mediums of 2%Ultroser G, 2mM/L L-Glu；
2. the DMEM culture mediums of 10%KnockOut Serum Replacement, 2mM/L L-Glu；
3. the DMEM culture mediums of 10%FBS, 2mM/L L-GLu.
Test procedure：The deciduous teeth of fresh peeling are digested using the separation method of Masako Miura etc. with collagenase type I
Afterwards, filter centrifugation obtains deciduous teeth mescenchymal stem cell and carries out original cuiture；Primary culture medium uses serum free medium I and no blood
Clear II isometric mixed culture medium of culture medium, cultivation cycle 7-14 days, after original cuiture by culture have deciduous teeth between fill
The square vase of matter stem cell is transferred to from carbon dioxide incubator in Biohazard Safety Equipment；Original culture medium is abandoned in suction, and it is slow that phosphate is added
Solution (DPBS) is rushed to washed once；DPBS solution is abandoned in suction, and the digestion 4 minutes of 37 DEG C of trypsase is added, and then cell is resuspended in DPBS,
And cell suspension is divided equally into 3 centrifuge tubes and is centrifuged, 400G room temperatures centrifuge 5 minutes；After centrifugation, it is separately added into 3 kinds of trainings
Foster base is cultivated；The adherent situation of cell is observed after 4 hours, and is photographed to record.
Interpretation of result：
(1) it makes discovery from observation, after cultivating 4 hours, the cell 1. 3. cultivated with culture medium using culture medium is in its square vase
The cell adhered to is observed in bottom, and largely adherent using 1. cell that culture medium is cultivated, and adherent rate, which is higher than, to be made
3. it is the cell of traditional fetal calf serum culture with culture medium, and the deciduous teeth mescenchymal stem cell 2. cultivated using culture medium, not
Any attached cell is observed, as shown in Fig. 1,2 and 3.
(2) after cultivating 18 hours, the adherent of the deciduous teeth mescenchymal stem cell cultivated in 3 kinds of culture mediums and life are observed again
Long situation.The cell 1. 3. cultivated with culture medium using culture medium is all adherent, and the cell 2. cultivated using culture medium is only few
It measures adherent.At this point, the cell 3. cultivated compared with culture medium of the cell 1. cultivated of culture medium compared to longer and narrower, shows the spindle of standard
Shape, as shown in Figure 4.
(3) in culture medium cultivating system 2., it is dry thin that the growth pattern of deciduous teeth mescenchymal stem cell is more likely to embryo
Born of the same parents, cell is slowly adherent, and adherent individual cells are proliferated to form clone, connects into piece with rear clone, cell arrangement is close, form
It is very long and narrow.This serum substitutes of KnockOut Serum Replacement are originally for cultivating embryonic stem cell and induction
Multipotential stem cell, in incubation, it requires both cells all in advance with the presence of feeder cells, therefore with culture
2. base cultivates deciduous teeth mescenchymal stem cell 6 days after, because cell is adherent poor, the degree of converging of cell is less than 30%, slow-growing
(as shown in Figure 5).Experiments have shown that KnockOut Serum Replacement are unfavorable for the adherency of deciduous teeth mescenchymal stem cell, but energy
The more preferable multipotency for maintaining stem cell, inhibits differentiation.
(4) with the increase of culture generation, the advantage that the rush cell adherence of the stem cell after 3 generations is 1. cultivated with culture medium is gradual
It disappears, proliferative capacity gradually lags behind the cell of fetal calf serum culture, and greatly variation occurs in cellular morphology, is no longer stem cell
Distinctive long and narrow, but shorten, cell that is partially round and having many feelers, and the cell 3. cultivated using culture medium, form
It is in still fusiform, as shown in Figure 6.
Embodiment 1：Deciduous teeth dental pulp mesenchymal stem cell serum-free culture
Serum-free complete medium I：Basal medium containing DMEM, 2mM/L L-Glutamines, 2%Ultroser G are molten
Liquid mixes well, and 4 DEG C of refrigerators preserve.
Serum-free complete medium II:Basal medium containing DMEM, 2mM/L L-Glutamines, 10%knockout SR
Serum substitute, 10ng/ml EGF, 10ng/ml bFGF.
Cultural method：
1) deciduous teeth of fresh peeling using Masako Miura etc. separation method, after being digested with collagenase type I, filter from
The heart obtains mescenchymal stem cell and carries out original cuiture, and original cuiture is complete using Serum-free complete medium I and serum free medium
Complete II it is isometric mixed after medium culture, cultivation cycle 7-14 days；
2) there is P0 to be abandoned for the cell original culture medium suction in the T75 square vases of deciduous teeth mescenchymal stem cell culture, be added
7.5ml DPBS washed once；
3) trypsin digestion of 0.8ml 0.25% is added 4 minutes, with Serum-free complete medium I weights after digestion
Outstanding cell；
4) cell suspension is transferred in sterile centrifugation tube, 400G, 20 DEG C, is centrifuged 5 minutes；
5) supernatant is removed after centrifuging, with Serum-free complete medium I with 4 × 104Cells/ml is inoculated in new T25 square vases
In, total 5ml；
6) square vase is positioned in carbon dioxide incubator, is cultivated 6 hours in 37 DEG C, 5% gas concentration lwevel；
7) square vase is transferred to from carbon dioxide incubator in Biohazard Safety Equipment, the sterile serum-free removed in culture bottle is complete
Full culture medium 1, is added the complete medium II of equivalent therewith, continues culture until cell confluency degree reaches 80%-90%；
8) secondary culture of a new round is carried out.
According to after 3 generation of aforesaid way culture with use 10%FBS DMEM medium culture primary cells after secondary culture 3
Detection in terms of the deciduous teeth mescenchymal stem cell progress form of a generation and phenotype marker expression.
Interpretation of result：
(1) after being cultivated 6 hours with culture medium I, deciduous teeth mescenchymal stem cell is all adherent, but adherent rear cellular morphology is simultaneously
Off-gauge fusiform, but stretched out many feelers, this fully shows cell and square vase bottom there are many anchored sites,
It is related that this is advantageous to cell adherence with Ultroser G, as shown in Figure 7.
(2) after cultivating for 3 generations, as it can be observed in the picture that cell shows the fusiform of standard, and arrangement mode shows stem cell
Distinctive swirl shape, and is applied alone the culture medium of the G containing Ultroser or the training of the Replacement of Serum containing KnockOut is applied alone
The cell for supporting base culture is compared, and is showed on cellular morphology, degree of converging more excellent.
2 deciduous teeth dental pulp mesenchymal stem cell serum-free culture of embodiment
Culture medium I and medium ii are mixed to prepare medium ii I：Basal medium containing DMEM, 2mM/L L- glutamy
Amine, 1%Ultroser G solution, 5%KnockOut SR serum substitutes, 10ng/ml EGF, 10ng/ml bFGF are fully mixed
Even, 4 DEG C of refrigerators preserve.
Cultural method：
1) deciduous teeth of fresh peeling using Masako Miura etc. separation method, after being digested with collagenase type I, filter from
The heart obtains mescenchymal stem cell and carries out original cuiture, and original cuiture is cultivated using medium ii I, culture cultivation cycle 7-14
It；
2) there is P0 to be abandoned for the cell original culture medium suction in the T75 square vases of deciduous teeth mescenchymal stem cell culture, be added
7.5ml DPBS washed once；
3) cell is resuspended with Serum-free complete medium III after 4 minutes in the trypsin digestion that 0.8ml 0.25% is added；
4) cell suspension is transferred in sterile centrifugation tube, 400G, 20 DEG C, is centrifuged 5 minutes；
5) supernatant is removed after centrifuging, using Serum-free complete medium III with 4 × 104Cells/ml is inoculated in the new side T25
In bottle, total 5ml, 37 DEG C of carbon dioxide incubator, 5% gas concentration lwevel, degree of mixing of the culture up to cell in saturated humidity
Up to 80%-90%；
6) it is passed after carrying out original cuiture according to after 3 generation of aforesaid way culture with the DMEM medium cultures of 10%FBS are used
Detection in terms of the deciduous teeth mescenchymal stem cell progress form for foster 3 generations of being commissioned to train and phenotype marker expression.
Interpretation of result：
After 3 generations of continuous passage culture, under inverted microscope observe cell form and growth conditions it is found that on
The deciduous teeth mescenchymal stem cell of cultural method culture and the deciduous teeth mescenchymal stem cell of traditional fetal calf serum culture are stated in cell
No significant difference in form, referring to Figure 10.It is the fusiform of standard, cell growth arrangement mode is swirl shape.Fluidic cell
Testing result shows the two no significant difference in terms of the expression of phenotype marker, is shown in Table 1.
Embodiment 3：Deciduous teeth dental pulp mesenchymal stem cell serum-free culture
Serum-free complete medium I：Basal medium containing DMEM, 2mM/L L-Glutamines, 4%Ultroser G are molten
Liquid mixes well, and 4 DEG C of refrigerators preserve.
Serum-free complete medium II:Basal medium containing DMEM, 2mM/L L-Glutamines, 10%KnockOut SR
Serum substitute, 10ng/ml EGF, 10ng/ml bFGF.
Cultural method：
1) deciduous teeth of fresh peeling using Masako Miura etc. separation method, after being digested with collagenase type I, filter from
The heart obtains mescenchymal stem cell and carries out original cuiture, and culture medium is that 4ml Serum-free complete mediums II and 1ml serum-frees are trained completely
Support the mixed culture medium of base I, cultivation cycle 7-14 days；
2) there is P0 to be abandoned for the cell original culture medium suction in the T75 square vases of deciduous teeth mescenchymal stem cell culture, 5ml is added
DPBS washed once；
3) cell is resuspended with Serum-free complete medium II after 4 minutes in the trypsin digestion that 0.8ml 0.25% is added；
4) cell suspension is transferred in sterile centrifugation tube, 400G, 20 DEG C, is centrifuged 5 minutes；
5) supernatant is removed after centrifuging, using Serum-free complete medium II with 5 × 104Cells/ml is inoculated in the new side T25
In bottle, it is inoculated with 4ml altogether, then adds 1ml culture mediums I, blows and beats mixing repeatedly with pipette, square vase is then positioned over dioxy
Change in carbon incubator, the culture in 37 DEG C, 5% gas concentration lwevel is until cell confluency degree is 80%-90%；
6) when cell reaches above-mentioned degree of converging, square vase is transferred to from carbon dioxide incubator in Biohazard Safety Equipment, is inhaled
Original culture medium is abandoned, 5ml DPBS are added and washed once, abandon DPBS；
7) 37 DEG C of 0.4ml trypsase is added to digest 4 minutes；
8) cell is resuspended with Serum-free complete medium II after digesting, and cell suspension is transferred to sterile centrifugation tube
In, 400G, centrifuges 5 minutes, supernatant is removed after centrifugation by 20 DEG C；
9) use Serum-free complete medium II with 5 × 104Cells/ml is inoculated in new square vase, is inoculated with 3.8ml altogether, so
After add 1.2ml culture mediums I, blow and beat mixing repeatedly with pipette, then square vase be positioned in carbon dioxide incubator,
37 DEG C, culture in 5% gas concentration lwevel is until cell confluency degree is 80%-90%；
10) when cell reaches above-mentioned degree of converging again, square vase is transferred to Biohazard Safety Equipment from carbon dioxide incubator
In, original culture is abandoned in suction, and 5ml DPBS are added and washed once, abandon DPBS；
11) it is added after the digestion 4 minutes of 37 DEG C of 0.4ml trypsase and cell is resuspended with Serum-free complete medium II, and will
Cell suspension is transferred in sterile centrifugation tube, 400G, 20 DEG C, centrifuges 5 minutes, supernatant is removed after centrifugation；
12) use Serum-free complete medium II with 5.7 × 104Cells/ml is inoculated in new square vase, is inoculated with altogether
Then 3.5ml adds 1.5ml culture mediums I, blown and beaten repeatedly with pipette and square vase is positioned over carbon dioxide incubator after mixing
In, the culture in 37 DEG C, 5% gas concentration lwevel is until cell confluency degree is 80%-90%.
Experimental result：
After cultivating three generations of cell, cellular morphology, growth pattern and medium culture 3 of the use containing fetal calf serum
The form of cell is not different after generation, as shown in figure 11.In addition, it is thin that the training method of above-described embodiment 3 will be used to harvest
The cell of medium culture of the use of born of the same parents and harvest containing fetal calf serum carries out surface marker expression analysis, it is known that is marked on surface
Above-mentioned three kinds of training methods do not have difference with fetal calf serum cultivating system in terms of the expression of will object, as shown in table 1.
The testing result for the cell surface marker that the different training methods of table 1 are harvested
| CD90 | CD73 | CD105 | CD34 | |
| Embodiment 1 | 99.8% | 99.82% | 90% | 0.16% |
| Embodiment 2 | 99.99% | 100% | 90% | 0.12% |
| Embodiment 3 | 99.99% | 99.99% | 89.88% | 2.29% |
| Use the training method of fetal calf serum | 99.99% | 100% | 88.76% | 0.08% |
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
Invention is explained in detail, it will be understood by those of ordinary skill in the art that：It still can be to aforementioned each implementation
Technical solution recorded in example is modified or equivalent replacement of some of the technical features；And these modification or
It replaces, the spirit and scope for various embodiments of the present invention technical solution that it does not separate the essence of the corresponding technical solution.
Claims (9)
1. a kind of Serum-free complete medium, which is characterized in that including successively or following two culture mediums for being used in mixed way：
Wherein, culture medium I is that addition L-Glutamine, 1%-4% can effectively facilitate cell adherence and expansion in basal medium
The serum substitute of exhibition, a concentration of 2mM/L of L-Glutamine；
Medium ii is that addition L-Glutamine, 7%-10% are used to cultivate ESC in basal medium and the serum of iPSC replaces
For object, a concentration of 2mM/L of L-Glutamine.
2. Serum-free complete medium according to claim 1, which is characterized in that serum substitute in the culture medium I
For Ultroser G.
3. Serum-free complete medium according to claim 1, which is characterized in that serum substitute in the medium ii
For KnockOut Serum Replacement.
4. Serum-free complete medium according to claim 1, which is characterized in that the basal medium be selected from α-MEM,
DMEM, DMDM/F12 or IMEM/F12.
5. Serum-free complete medium according to claim 1, which is characterized in that further include in the medium ii EGF,
bFGF。
6. application of the claim 1-5 any one of them culture medium in cultivating mescenchymal stem cell.
7. a kind of culture method in serum-free of deciduous teeth mescenchymal stem cell, which is characterized in that the method uses such as claim 1
Stem cell of the mixed culture medium culture of culture medium I and the medium ii per generation, culture medium I wherein in mixed culture medium
Serum substitute ratio with medium ii is 1:1-1:5.
8. a kind of culture method in serum-free of deciduous teeth mescenchymal stem cell, which is characterized in that the described method comprises the following steps:
A, after being incubated the cell 4h-24h after passage using culture medium I as described in claim 1, this kind of culture medium is removed；
B, continue culture to cell confluency degree using medium ii as described in claim 1 and reach 80%-90%.
C, it passes on every time all with above-mentioned training method.
9. a kind of culture method in serum-free of deciduous teeth mescenchymal stem cell, which is characterized in that the method uses such as claim 1
The proportional region of the mixed culture medium of the culture medium I and medium ii, culture medium I and medium ii is 1:5-3:7, and with
The increase for culture generation, the ratio of culture medium I is to gradually increase in mixed culture medium.
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| CN109439617A (en) * | 2018-12-25 | 2019-03-08 | 成都赋智健康科技有限公司 | A kind of stem cell serum-free culture medium and preparation method thereof |
| CN110642956A (en) * | 2019-08-14 | 2020-01-03 | 浙江万寿康药业有限公司 | Extraction and application of dendrobium officinale O-acetyl glucomannan |
| CN111172105A (en) * | 2020-01-17 | 2020-05-19 | 中国人民解放军联勤保障部队第九二〇医院 | Preparation method of mesenchymal stem cell secretin |
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| CN106947736A (en) * | 2017-04-14 | 2017-07-14 | 青岛青春派生物科技有限公司 | A kind of human adipose mesenchymal stem cells Serum-free complete medium |
| CN107236702A (en) * | 2017-05-24 | 2017-10-10 | 北京再生生物科技研究院有限公司 | A kind of preparation method of perinatal period Mesenchymal Stem Cells from Umbilical Cord master cell bank |
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