CN104120105A - Serum-free medium used for culture of elderly people cartilage cells - Google Patents
Serum-free medium used for culture of elderly people cartilage cells Download PDFInfo
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- CN104120105A CN104120105A CN201310150092.7A CN201310150092A CN104120105A CN 104120105 A CN104120105 A CN 104120105A CN 201310150092 A CN201310150092 A CN 201310150092A CN 104120105 A CN104120105 A CN 104120105A
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Abstract
The invention discloses a serum-free medium used for culture of elderly people cartilage cells, the-free medium comprises a basal medium and an additive comprising the components of the following concentration: 233muM-287 muM of vitamin C, 3.7mu M-8.9 muM of linoleic acid, 9muM-17 muM of cholesterol, 6nM-15 nM of dexamethasone, 43 muM-58mu M of acetyl cysteine, 18 mug / mL-32 mu g / mL of transferrin, 22nM-52 nM of sodium selenite, 13 muM-24 muM of sodium pantothenate, 28 muM-43 muM of biotin, 7 mug / mL-18 mu g / mL of insulin, 2 ng / mL-9 ng / mL of epidermal growth factor, 2 ng / mL-9 ng / mL of fiber growth factor, 2 ng / mL-9 ng / mL of platelet derived growth factor, and 0.5%-2.5% of human serum albumin. The cell culture time is short, and the cell biological performance is good.
Description
Technical field
The present invention relates to the serum free medium that a kind of vitro culture human articular chondrocytes is used, particularly a kind of for cultivating older population chondrocyte's serum free medium.
Background technology
Articular cartilage damage, is that the damage that wound or pathology reason cause all can not spontaneously be repaired, and all can causes the degeneration of cartilage conventionally, and then cause osteoarthritis.Effective alleviating pain for the elderly, recover the method for knee joint function and only have artificial knee joint replacement, although this method to a certain extent can alleviating pain symptom, but the operation method of similar " invasion formula " and the finite life of joint prosthesis, all can make patient's function of joint can not get permanent maintaining.
At present, apply autologous chondrocyte cell transplantation (ACI) technology and can treat the knee joint surface cartilage defect of this type of traumatic (or osteochondritis dissecans), in recent years, people also utilize Bioengineered timbering material as cell, to transport the advantage of carrier, the arthroscope equipment that exploitation makes new advances, and be applied in ACI technology, but no matter in experimentation on animals or in clinical case, the application of ACI all will have been done strict restriction the age, and old animal or patient (age is greater than 55 years old patient) are excluded outside treatment.In fact take from the cartilaginous tissue in the non-heavy burden of gerontal patient district, chondrocyte through in-vitro separation amplification cultivation, its multiplication capacity and differentiation capability are all far away from youngster, have at present gerontal patient's quantity of joint cartilage aspect Treatment need huge, but existing feasible cell therapy means all can not effectively meet this demand.
In addition; traditional chondrocyte's condition of in vitro culture---using autoserum or bovine serum (research is used) as substratum added ingredients; because the cartilaginous tissue in human body is not survive in the situation that there is no blood confession; only dependent cells autocrine or paracrine factor carry out maintenance metabolism balance, so usually can cause the non-physiological response of culturing cell.
Summary of the invention
The object of this invention is to provide a kind of for cultivating older population chondrocyte's serum free medium.It can make the chondrocyte in aging donor cartilaginous tissue source on the basis without any serum of interpolation, can break up by Effective multiplication in a short time, and the biological characteristics that can keep chondrocyte, meet the clinical application of the aged people of One's name is legion, make its function of joint obtain permanent maintaining.
Technical scheme of the present invention: a kind ofly comprise basic medium and additive for cultivating older population chondrocyte's serum free medium, basic medium is DMEM/F12 (1:1), and the composition of this additive, in final concentration, comprising:
The vitamins C of 233 μ M ~ 287 μ M,
The linolic acid of 3.7 μ M ~ 8.9 μ M,
The cholesterol of 9 μ M ~ 17 μ M,
The dexamethasone of 6nM ~ 15nM,
The acetylcysteine of 43 μ M ~ 58 μ M,
The Transferrins,iron complexes of 18 μ g/mL ~ 32 μ g/mL,
The Sodium Selenite of 22nM ~ 52nM,
The sodium pantothenate of 13 μ M ~ 24 μ M,
The vitamin H of 28 μ M ~ 43 μ M,
The Regular Insulin of 7 μ g/mL ~ 18 μ g/mL,
The Urogastron of 2 ng/mL ~ 9ng/mL
The fibroblast growth factor of 2 ng/mL ~ 9ng/mL
The Thr6 PDGF BB of 2 ng/mL ~ 9ng/mL,
0.5% ~ 2.5% human serum albumin.
Aforesaid for cultivating older population chondrocyte's serum free medium, the composition of described additive, in final concentration, comprising:
The vitamins C of 250 μ M,
The linolic acid of 4.5 μ M,
The cholesterol of 13 μ M,
The dexamethasone of 10nM,
The acetylcysteine of 50 μ M,
The Transferrins,iron complexes of 25 μ g/mL,
The Sodium Selenite of 30nM,
The sodium pantothenate of 17 μ M,
The vitamin H of 33 μ M,
The Regular Insulin of 10 μ g/mL,
The Urogastron of 5ng/mL.
The fibroblast growth factor of 5ng/mL,
The Thr6 PDGF BB of 5 ng/mL,
1% human serum albumin.
Substratum concrete preparation process as follows:
1) configure base substratum DMEM/F12 (1:1).
2) under aseptic condition, additive is joined in basic medium, thereby make substratum be applicable to the articular chondrocytes of the old donor source of vitro culture.
3) substratum, before putting into chondrocyte, first carries out 37 ° of water-baths approximately 30 minutes, in cell cultivation process, keeping the temperature of substratum, is 37 °, 5%CO2 concentration, and other steps are identical with common cultured chondrocytes step.
Contriver is in invention process, having prepared in addition portion adds the basic medium of foetal calf serum (FBS) as experiment contrast group, experimental result shows in identical culture cycle, and additive of the present invention is promoting cell proliferation, remains more remarkable in the effect of cellular form.In addition, also prepared the old donor serum free medium as preferred control group, result shows in identical culture cycle, and preferred version of the present invention is promoting cell proliferation, remains more remarkable in the effect of cellular form.
In addition, take cell amplification to self 5 times of times that spent be comparison other, contriver has also prepared young donor serum-free culture experiment contrast group, the preferred old donor serum free medium of the present invention, conduct preferably old donor serum free medium, old donor 10% foetal calf serum of control group, experimental result shows, as the chondrocyte in preferred old donor serum free medium, increase 5 times of needed times, approach very much young donor and increase 5 times of needed times, be obviously less than two other experiment contrast body.Preferred version of the present invention is best in the effect aspect incubation time, acquisition cell aspect of performance, maintenance cellular form.
In old donor serum free medium of the present invention, add SF additive, cultivate for some time, then do cartilage specificity gene test, result shows that SF additive does not change cellularity, and does not hinder the expression of its specific gene.
Substratum of the present invention has been selected novel for cultivating older population chondrocyte's serum free medium, this substratum can break up by Effective multiplication in a short time, can keep chondrocyte's biological characteristics again, therefore can meet the clinical application of the aged people of One's name is legion, make its function of joint obtain permanent maintaining.Large-scale promotion application is convenient in the present invention.
Accompanying drawing explanation
Fig. 1 is the chondrocyte figure that adds the base culture base of foetal calf serum (FBS);
Fig. 2 is the chondrocyte figure of the culture medium culturing of preferred version of the present invention;
Fig. 3 is that the present invention is as the chondrocyte figure of the culture medium culturing of preferred control group;
Fig. 4 is the time comparison diagram of preferred version of the present invention and experiment contrast group cultured cartilage cell;
Fig. 5 is the chondrocyte's that cultivates of the present invention cartilage specificity gene test figure.
Embodiment
Below in conjunction with drawings and Examples, the present invention is further illustrated, but not as the foundation to the present invention's restriction.
Embodiment.For cultivating older population chondrocyte's a serum free medium, comprise basic medium and additive, basic medium is DMEM/F12 (1:1), the composition of this additive, in final concentration, comprising:
The vitamins C of 233 μ M ~ 287 μ M,
The linolic acid of 3.7 μ M ~ 8.9 μ M,
The cholesterol of 9 μ M ~ 17 μ M,
The dexamethasone of 6nM ~ 15nM,
The acetylcysteine of 43 μ M ~ 58 μ M,
The Transferrins,iron complexes of 18 μ g/mL ~ 32 μ g/mL,
The Sodium Selenite of 22nM ~ 52nM,
The sodium pantothenate of 13 μ M ~ 24 μ M,
The vitamin H of 28 μ M ~ 43 μ M,
The Regular Insulin of 7 μ g/mL ~ 18 μ g/mL,
The Urogastron of 2 ng/mL ~ 9ng/mL,
The fibroblast growth factor of 2 ng/mL ~ 9ng/mL,
The Thr6 PDGF BB of 2 ng/mL ~ 9ng/mL,
0.5% ~ 2.5% human serum albumin.
1. 1 kinds of embodiment, for cultivating older population chondrocyte's serum free medium, comprise basic medium and additive, and basic medium is DMEM/F12 (1:1), and the composition of this additive, in final concentration, comprising:
The vitamins C of 250 μ M,
The linolic acid of 4.5 μ M,
The cholesterol of 13 μ M,
The dexamethasone of 10nM,
The acetylcysteine of 50 μ M,
The Transferrins,iron complexes of 25 μ g/mL,
The Sodium Selenite of 30nM,
The sodium pantothenate of 17 μ M,
The vitamin H of 33 μ M,
The Regular Insulin of 10 μ g/mL,
The Urogastron of 5ng/mL.
The fibroblast growth factor of 5ng/mL,
The Thr6 PDGF BB of 5 ng/mL,
1% human serum albumin.
2. 1 kinds of embodiment, for cultivating older population chondrocyte's serum free medium, comprise basic medium and additive, and basic medium is DMEM/F12 (1:1), and the composition of this additive, in final concentration, comprising:
The vitamins C of 240 μ M,
The linolic acid of 4 μ M,
The cholesterol of 15 μ M,
The dexamethasone of 10nM,
The acetylcysteine of 45 μ M,
The Transferrins,iron complexes of 20 μ g/mL,
The Sodium Selenite of 25nM,
The sodium pantothenate of 18 μ M,
The vitamin H of 35 μ M,
The Regular Insulin of 10 μ g/mL,
The Urogastron of 5ng/mL,
5ng/mL fibroblast growth factor,
5ng/mL Thr6 PDGF BB,
1% human serum albumin.
Above in each embodiment, the substratum that DMEM/F12 (1:1) Shi You GIBCO company produces, production number: 11330057.
In above-mentioned additive component,
The vitamins C of 233 μ M ~ 287 μ M, for maintaining the biologically active substance of Growth of Cells, rises and regulates and control action kou in cellular metabolism, maintains chondrocyte's phenotype, Promote cell's growth.
The cholesterol of the linolic acid of 3.7 μ M ~ 8.9 μ M, 9 μ M ~ 17 μ M is used for maintaining cell eubolism.
The dexamethasone of 6nM ~ 15 nM, is adrenocortical hormone, for Promote cell's growth, maintains functionally active.
The acetylcysteine of 43 μ M ~ 58 μ M, for promoting emiocytosis Type Ⅱ collagen, prevents cell senescence.
The Transferrins,iron complexes of 18 μ g/mL ~ 32 μ g/mL, provides ferro element, for Promote cell's growth.
The Sodium Selenite of 22nM ~ 52nM, for Promote cell's growth.
The sodium pantothenate of 13 μ M ~ 24 μ M, for improving tethelin, Promote cell's growth.
The vitamin H of 28 μ M ~ 43 μ M, for Promote cell's growth.
The Regular Insulin of 7 μ g/mL ~ 18 μ g/mL, for promoting chondrocyte proliferation, cell matrix secretion.
The Urogastron of 2 ng/mL ~ 9ng/mL, for promoting chondrocyte proliferation and promoting cell matrix secretion.
The fibroblast growth factor of 2 ng/mL ~ 9ng/mL, for promoting chondrocyte proliferation, promotes cell matrix secretion.
The Thr6 PDGF BB of 2 ng/mL ~ 9ng/mL, for promoting chondrocyte proliferation, promotes cell matrix secretion.
0.5% ~ 2.5% human serum albumin, for Promote cell's growth.
Above in each embodiment, substratum concrete preparation process as follows:
1) configure base substratum DMEM/F12 (1:1).
2) under aseptic condition, additive is joined in basic medium, thereby make substratum be applicable to the articular chondrocytes of the old donor source of vitro culture.
3) substratum, before putting into chondrocyte, first carries out 37 ° of water-baths approximately 30 minutes, in cell cultivation process, keeping the temperature of substratum, is 37 °, 5%CO2 concentration, and other steps are identical with common chondrocyte's step.
Contriver is in invention process, having prepared in addition portion adds the basic medium of foetal calf serum (FBS) as experiment contrast group, experimental result as shown in Figure 1 and Figure 2, we can find in identical culture cycle, additive of the present invention is promoting cell proliferation, remains more remarkable in the effect of cellular form.In addition, also prepared the old donor serum free medium as preferred control group, comparison diagram 2, Fig. 3, we can find in identical culture cycle, preferred version of the present invention is promoting cell proliferation, remains more remarkable in the effect of cellular form.
In addition, take cell amplification to self 5 times of times that spent be comparison other, contriver has also prepared young donor serum-free culture experiment contrast group, the preferred old donor serum free medium of the present invention, old donor serum free medium as preferred control group, old donor 10% foetal calf serum, experimental result as shown in Figure 4, we can find, as the chondrocyte in preferred old donor serum free medium, increase 5 times of needed times, approaching very much young donor increases 5 times of needed times, obviously be less than two other experiment contrast body.Preferred version of the present invention is best in the effect aspect incubation time, acquisition cell aspect of performance, maintenance cellular form.
In old donor serum free medium of the present invention, add SF additive, cultivate for some time, then do cartilage specificity gene test, result as shown in Figure 5, shows that SF additive does not change cellularity, and does not hinder the expression of its specific gene.
Claims (3)
1. for cultivating older population chondrocyte's a serum free medium, it is characterized in that, comprise basic medium and additive, basic medium is DMEM/F12 (1:1), and the composition of this additive, in final concentration, comprising:
The vitamins C of 233 μ M ~ 287 μ M,
The linolic acid of 3.7 μ M ~ 8.9 μ M,
The cholesterol of 9 μ M ~ 17 μ M,
The dexamethasone of 6nM ~ 15 nM,
The acetylcysteine of 43 μ M ~ 58 μ M,
The Transferrins,iron complexes of 18 μ g/mL ~ 32 μ g/mL,
The Sodium Selenite of 22nM ~ 52 nM,
The sodium pantothenate of 13 μ M ~ 24 μ M,
The vitamin H of 28 μ M ~ 43 μ M,
The Regular Insulin of 7 μ g/mL ~ 18 μ g/mL,
The Urogastron of 2 ng/mL ~ 9 ng/mL,
The fibroblast growth factor of 2 ng/mL ~ 9 ng/mL,
The Thr6 PDGF BB of 2 ng/mL ~ 9 ng/mL,
0.5% ~ 2.5% human serum albumin.
2. according to claim 1ly for cultivating older population chondrocyte's serum free medium, it is characterized in that, described additive component, in final concentration, comprising:
The vitamins C of 250 μ M,
The linolic acid of 4.5 μ M,
The cholesterol of 13 μ M,
The dexamethasone of 10nM,
The acetylcysteine of 50 μ M,
The Transferrins,iron complexes of 25 μ g/mL,
The Sodium Selenite of 30nM,
The sodium pantothenate of 17 μ M,
The vitamin H of 33 μ M,
The Regular Insulin of 10 μ g/mL,
The Urogastron of 5ng/mL.
The fibroblast growth factor of 5ng/mL,
The Thr6 PDGF BB of 5 ng/mL,
1% human serum albumin.
3. the purposes of serum free medium according to claim 1 and 2, is characterized in that, it is used to cultivate older population chondrocyte.
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| CN201310150092.7A CN104120105A (en) | 2013-04-26 | 2013-04-26 | Serum-free medium used for culture of elderly people cartilage cells |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255814A (en) * | 2015-10-27 | 2016-01-20 | 上海科医联创生物科技有限公司 | Chondrocyte induction substrate for microfracture surgery and preparation method |
| CN106459912A (en) * | 2014-11-14 | 2017-02-22 | 再生医科学股份有限公司 | Method for serum-free culture of cartilage cells and serum-free culture medium |
| CN106520685A (en) * | 2016-12-21 | 2017-03-22 | 江西宜信堂医疗科技有限公司 | Serum-free medium applicable to chondrocyte in vitro culture and preparation method of serum-free medium |
-
2013
- 2013-04-26 CN CN201310150092.7A patent/CN104120105A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106459912A (en) * | 2014-11-14 | 2017-02-22 | 再生医科学股份有限公司 | Method for serum-free culture of cartilage cells and serum-free culture medium |
| CN106459912B (en) * | 2014-11-14 | 2021-05-25 | 再生医科学股份有限公司 | Serum-free culture method and serum-free culture medium for chondrocytes |
| US11427808B2 (en) | 2014-11-14 | 2022-08-30 | Regenesis Science Co., Ltd. | Method for serum-free culture of chondrocytes and serum-free culture medium |
| CN105255814A (en) * | 2015-10-27 | 2016-01-20 | 上海科医联创生物科技有限公司 | Chondrocyte induction substrate for microfracture surgery and preparation method |
| CN106520685A (en) * | 2016-12-21 | 2017-03-22 | 江西宜信堂医疗科技有限公司 | Serum-free medium applicable to chondrocyte in vitro culture and preparation method of serum-free medium |
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Application publication date: 20141029 |