CN102399742B - Serum-free culture fluid for culture of salivary gland epidermal cell and salivary gland stem cell of mammals - Google Patents
Serum-free culture fluid for culture of salivary gland epidermal cell and salivary gland stem cell of mammals Download PDFInfo
- Publication number
- CN102399742B CN102399742B CN201110303107.XA CN201110303107A CN102399742B CN 102399742 B CN102399742 B CN 102399742B CN 201110303107 A CN201110303107 A CN 201110303107A CN 102399742 B CN102399742 B CN 102399742B
- Authority
- CN
- China
- Prior art keywords
- serum
- cell
- salivary gland
- sialisterium
- mammals
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention provides serum-free culture fluid for culture of salivary gland epidermal cells and salivary gland stem cells of mammals, which is prepared by using MCDB153HAA as basic culture medium and adding amino acid, vitamins, salts, lipid, trace elements, buffer fluid, hormone-like compounds, transgenic metalloprotein, antioxidants, seralbumin, glucide, purine, pyrimidine base substances and pH value indicators. The serum-free culture fluid has the advantages that salivary gland epidermal cells of the mammals can vigorously grow, good cell activity and physiological properties are maintained, in addition, the serum-free culture fluid is also suitable for the salivary gland epidermal cell culture, is particularly suitable for the field of study relevant to biological tissue engineering and belongs to the commercial cell culture fluid.
Description
Technical field
The present invention relates to RESEARCH ON CELL-BIOLOGY field, relate in particular to Mammals sialisterium epithelial cell and sialisterium stem cell culture technique field.
Background technology
Sialisterium is one of mammiferous important exocrine gland, and its secretory product can help digest, protect mucous membrane and antibacterial bacteriostatic.The major function of sialisterium is produce and salivate, and under normal circumstances, people's salivation every day amount approximately 1 rises to 1.5 liters.Saliva 25% by parotid secretion, 5% is secreted by sublingual gland, 10% by various glandulae salivariae minoreses (labial gland, buccal glands, palatine gland etc.) secretion, 60% is secreted by glandula submandibularis.From animal and people's submaxillary gland, find or isolate nearly 30 kinds of biologically active polypeptidess at present, as nerve growth factor (NGF), Urogastron (EGF), endothelial growth stimulating factor (EGSF), erythropoietin (EPO), marrow clone stimulating factor (CSF), feritin (renin), kallikrein (kallikrein, kk), Somatostatin, Regular Insulin, amylase, acid phosphatase, rnase and ester peptase etc.These peptide materials or direct secretion enter blood, or enter digestive tube again by stomach and intestine absorbed into serum with saliva, and the physiological activity of Various Tissues and cell is played to important regulative.
The substantial part of sialisterium is comprised of acinus and conduit.Acinus is the secretory portion of body of gland, by monoptychic gland epithelial cell around forming.Conduit is the pipeline of excrete thing, ductal epithelial cell, consists of.Existing achievement in research demonstration, sialisterium stem cell is probably present in conduit position.Therefore sialisterium epithelial cell is the critical function cell that forms body of gland, secretion and excretion saliva, the epithelial dysfunction of sialisterium, will cause multiple disease of salivary gland: (1) with advancing age, the acinus part atrophy of body of gland, cause fibering change simply and adipocyte to increase, salivary flow reduces.It is a kind of alternative phenomenon after acinus atrophy that this adipocyte increases; (2) the ancient Li Zishi disease of rice, house Green's Cotard (dry syndrome).This is the autoimmune disorder of sialisterium, and lymphocytic infiltration also replaces acinus; (3) tumor of head and neck patient is after accepting radiation treatment, and sialisterium epithelial cell sustains damage, and the secretion of saliva also can significantly reduce, and affects patient's life quality; (4) the sialisterium epithelial cell formation tumour that cancerates, as pleomorphic adenoma, adenoid cystic carcinoma etc.Tumour cell affects the normal function of sialisterium, but the pathogeny of these diseases it be unclear that.Therefore, define the epithelial biological characteristics of sialisterium, can help in treatment, to avoid damaging normal sialisterium, and utilize sialisterium stem cell to repair salivary organization and function thereof.
Sialisterium epithelial cell is the important materials of research sialisterium, and vitro culture sialisterium epithelial cell and sialisterium stem cell are the important steps of this research field.In sialisterium epithelial cell nutrient solution in the past, be added with serum.Complicated ingredient in serum has been covered the epithelial genuine property of sialisterium, and has accelerated the differentiation of sialisterium epithelial stem cell, and cell cannot long-term cultivation and subculture.Sold on the market in recent years the superficial cell serum-free medium of import, and had minority report prompting, this nutrient solution can be cultivated sialisterium epithelial cell, and can subculture 3-4 time.The people such as Lombaert in 2008 have reported with serum-free cell culture medium and have cultivated mouse submandibular gland epithelial cell (I.M.Lombaert, J.F.Brunsting, P.K.Wierenga, H.H.Kampinga, G.de Haan and R.P.Coppes, Keratinocyte growth factor prevents radiation damage to salivary glands by expansion of the stem/progenitor pool, Stem Cells 26 (2008), 2595-2601), they have added epithelical cell growth factor EGF (20ng/mL) in DMEM/F12 basic medium, fibroblast growth factor FGF2 (20ng/mL), commercial prod N2 (1/100), Regular Insulin (10 μ g/mL) and dexamethasone (1 μ M).N2 is a kind of commercial prod, and its component is indefinite, and expensive.EGF and FGF2 are also high price commodity, so this area is strong in the urgent need to a kind of with low cost, applicability, the serum-free cell culture medium that is applicable to sialisterium epithelial cell and stem cell.The epithelial stable growth of sialisterium in experimentation can be guaranteed on the one hand, on the other hand, sialisterium stem cell can be obtained.
Summary of the invention
Object of the present invention is just to provide a kind of serum-free medium and compound method and using method for Mammals sialisterium epithelial cell and the cultivation of sialisterium stem cell.
For achieving the above object, the invention provides a kind of serum-free medium, is in basic medium, to add amino acid, VITAMIN, salt, lipid, trace element, damping fluid, hormones compound, turn metalloprotein, antioxidant, serum albumin, carbohydrate, purine, pyrimidine bases material and pH pH indicator.
Basic medium is MCDB153HAA; Or three kinds of substratum that substratum MCDB153HAA substratum, DMEM in high glucose and RPMI1640 three mix; Concrete, can be in MCDB153HAA basic medium, the RPMI1640 substratum allotment of adding the DMEM in high glucose and 1/20 (v/v) of 1/20 (v/v) forms, and above-mentioned this substratum is a kind of substratum of applicable epithelial cell growth.
Amino acid is selected from one or more in L-Ala, arginine, l-asparagine acid, aspartic acid, halfcystine, Gelucystine, L-glutamic acid, glutamine, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine, α-amino-isovaleric acid, oxyproline; Add the preferred glutamine 600～900mg/L of content, arginine 100～300mg/L, leucine, Serine, halfcystine, α-amino-isovaleric acid, proline(Pro) or Methionin 20～100mg/L, Histidine, l-asparagine acid, Threonine, L-glutamic acid or glycine content are 10～20mg/L, the content of Isoleucine, tyrosine disodium, L-Ala, methionine(Met) or phenylalanine is 5～10mg/L, and the content of aspartic acid, tryptophane or oxyproline is 0.5～5mg/L;
More preferably amino acid and content are as follows:
VITAMIN is selected from one or more in vitamin H, choline chloride 60, folic acid, inositol, putrescine, calcium pantothenate, riboflavin, VITMAIN B1, vitamin B6, vitamin B12, Pyridoxine HCL, thiamine hydrochloride, niacinamide, amido M-nitro benzoic acid; Adding content is preferably as follows, inositol or choline chloride 60 content are 10～30mg/L, folic acid or putrescine 1～5mg/L, the content of amido M-nitro benzoic acid, pantothenic acid, VITMAIN B1, vitamin B6, vitamin B-12, niacinamide or riboflavin is 0.1～1mg/L, vitamin H 0.01～0.1mg/L;
More preferably VITAMIN and content are as follows:
Salt be selected from calcium chloride, sodium-chlor, Repone K, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic, Sodium.alpha.-ketopropionate, sodium acetate, anhydrous, water glass, nitrocalcite, in one or more; The following sodium-chlor 5000～7000mg/L of preferred content, sodium bicarbonate 1000～1500mg/L, AMSP, Sodium.alpha.-ketopropionate or sodium acetate, anhydrous 100～400mg/L, water glass 0.1～1mg/L, Repone K 200～500mg/L, calcium chloride or nitrocalcite 1～20mg/L;
More preferably salt and content are as follows:
Lipid be selected from oleic acid, linolic acid, monoethanolamine, one or more in Thioctic Acid; Preferred oleic acid 70～140mg/L wherein, monoethanolamine 0.1～1.5mg/L, Thioctic Acid 0.1～1.0mg/L; More preferably oleic acid 88～126mg/L, monoethanolamine 0.48～1.1mg/L, Thioctic Acid 0.15～0.2mg/L.
Trace element is selected from one or more in copper sulfate, iron nitrate, ferrous sulfate, magnesium chloride, magnesium sulfate, zinc sulfate, ammonium meta-vanadate, Sodium Selenite, molybdic acid, nickelous chloride, tin chloride, ferric sulfate, manganous sulfate; Preferred content is as follows, magnesium chloride 90～140mg/L, manganous sulfate, ferric sulfate 1～10mg/L, zinc sulfate 0.1～1mg/L, copper sulfate, iron nitrate or Sodium Selenite 0.001～0.01mg/L, nickelous chloride, ammonium meta-vanadate, molybdic acid 0.0001～0.0015mg/L, tin chloride 0.00001～0.00015mg/L;
More preferably trace element and content are as follows:
Damping fluid is sodium bicarbonate and/or HEPES; Preferred content is sodium bicarbonate 1000～1500mg/L, HEPES 6000～7000mg/L; More preferably content is sodium bicarbonate 1180～1210mg/L, HEPES6600～6700mg/L.
Hormones compound is Regular Insulin, preferably 1～20mg/L, more preferably 1.6～12.8mg/L;
Turning metalloprotein is Transferrins,iron complexes, preferably 3～8mg/L, more preferably 3.6～5.8mg/L;
Serum albumin is bovine serum albumin, preferably 400～700mg/L, more preferably 450～600mg/L;
Carbohydrate is glucose and Sodium.alpha.-ketopropionate, preferably glucose 3000～4000mg/L, pyruvic acid 45～57mg/L;
Antioxidant is beta-mercaptoethanol and/or gsh, preferred beta-mercaptoethanol 0.52～0.88mg/L, gsh 0.04～0.06mg/L;
Purines is VITAMIN B4, preferably 27～29mg/L;
Described pyrimidine bases material is thymus pyrimidine, preferably 0.6～0.75mg/L;
PH pH indicator is phenol red, preferably 8～9mg/L.
Preferred, the interpolation combination of above-mentioned nutrient solution is as follows:
Above-mentioned serum-free cell culture medium can be cultivated for Mammals sialisterium epithelial cell, also can be for the primary cell culture of other normal epithelial tissues, and preferred breast epithelium, pancreas epithelium, Keratoderma epithelium, mucous epithelium.
Further, can also add 12～15mg/L Medulla Bovis seu Bubali extract (BPE) in above-mentioned nutrient solution, BPE is the mixture of one kind of multiple somatomedins, from the hypophysis group of ox, extracts, and can effectively promote cell proliferation.The nutrient solution of above-mentioned acquisition, also can be for the primary cell culture of other normal epithelial tissues for better cultivating enrichment Mammals sialisterium epithelial cell, and normal epithelial is organized preferred breast epithelium, pancreas epithelium, Keratoderma epithelium, mucous epithelium etc.
The using method of serum-free cell culture medium of the present invention is Mammals sialisterium epithelial cell to be placed in to aforementioned serum-free medium cultivate; Or the tissue block of Mammals sialisterium epithelium is shredded or be digested to unicellular being inoculated in aforesaid serum-free medium carry out former culture, at 37 ℃, 5%CO
2under environment, cultivate after 48-72h, can obtain epithelial cell.
Or make Mammals normal epithelial histocyte be placed in described serum-free medium to cultivate, or the tissue block of mammalian epithelial tissue is shredded or be digested to unicellular being inoculated in described serum-free medium carry out former culture, at 37 ℃, 5%CO
2under environment, cultivate after 48-72h, can obtain epithelial cell.Normal epithelial tissue comprises: mammary epithelial cell, pancreas epithelium, Keratoderma epithelium, mucous epithelium etc.
Can select, in above-mentioned nutrient solution, not add BPE, further add people FGF2, heparin sodium, L-AA-2-tricresyl phosphate sodium salt and NSC 334200 dipeptides, for cultivating enrichment Mammals sialisterium stem cell.
Wherein, when NSC 334200 dipeptides is applied to the cultivation of sialisterium epithelial stem cell, NSC 334200 dipeptides is more stable under culture condition, not only can be continuously stem cell energy is provided, and can avoid the accumulation of the toxic metabolite that causes because of natural metabolism; L-AA-2-tricresyl phosphate the sodium salt adding is a kind of antioxidant more stable than vitamins C, and it can keep for a long time active in nutrient solution, greatly delays the speed of sialisterium epithelial stem cell aging, and contributes to maintain the characteristic of stem cell; Further added heparin sodium, to promote the signal transduction of FGF2 in cell.
Above-mentioned serum-free cell culture medium is used for cultivating sialisterium epithelial stem cell, concrete using method be the normal sialisterium epithelial stem cell of Mammals is placed in to above-mentioned acquisition serum-free medium at 37 ℃, 5%CO
2under environment, cultivate.
The present invention provides the compound method of above-mentioned serum-free medium on the other hand, comprises the following steps:
(1) take MCDB153HAA as basic medium, add amino acid, VITAMIN, salt, lipid, trace element, damping fluid, hormones compound, turn metalloprotein, antioxidant, serum albumin, carbohydrate, purine, pyrimidine bases material and pH pH indicator;
(2) add the Medulla Bovis seu Bubali extract (BPE) of 12～15mg/L in the resulting nutrient solution of step (1); Or add respectively L-AA-2-tricresyl phosphate sodium salt of 9.5～12.5mg/L, and the heparin sodium of 0.3～1mg/L, the NSC 334200 dipeptides of the FGF2 of 5～20 μ g/L and 400-460mg/L is to the resulting nutrient solution of step (1).
Concrete, can be that directly to select MCDB153HAA be basic medium, or in MCDB153HAA basic medium, add that the composition allotment of RPMI1640 substratum of the DMEM in high glucose and 1/20 (v/v) of 1/20 (v/v) forms.This substratum is a kind of substratum of applicable epithelial cell growth.
Next, in basic medium, add amino acid, VITAMIN, salt, lipid, trace element, damping fluid, serum albumin and prepare basic culture solution, potential of hydrogen is adjusted to 7.0～7.2, after filtration sterilising treatment; Add again Transferrins,iron complexes, Regular Insulin, monoethanolamine, Sodium Selenite, beta-mercaptoethanol obtain further nutrient solution;
Can be to the Medulla Bovis seu Bubali extract (BPE) that adds 12～15mg/L in nutrient solution; Or add respectively L-AA-2-tricresyl phosphate sodium salt of 9.5～12.5mg/L, and the heparin sodium of 0.3～1mg/L, the NSC 334200 dipeptides of the FGF2 of 5～20 μ g/L and 400-460mg/L is to nutrient solution obtained above.
Wherein, said components is dissolved in without (resistivity is 18.2 Ω) configuration in thermal source ultrapure water and forms.
Serum-free medium of the present invention can make cultured cells keep good multiplication capacity and biological characteristics.The positively effect of serum-free medium of the present invention is:
(1) not containing serum, experimental system is clear, composition is definite, is conducive to the analysis of experimental result, the unclear impact that experimental result is brought of Experimental Background of having avoided unknowable composition to cause;
(2), in serum-free medium of the present invention as add BPE, can greatly promote epithelial propagation and cell survival rate, for experiment provides sufficient cell quantity;
(3) applied widely, be applicable to the epithelial cultivation of polytype Mammals, as mammary epithelial cell, pancreas epithelial cell and superficial cell etc.;
(4) be applicable to sialisterium stem cell and cultivate, in 3-D Matrigel culture system, can break up and become sialisterium conduit or acinus.
Serum-free medium definite ingredients of the present invention, additive composition is simple and easy to preparation, and cost is cheaper, the wider model of range of application.The present invention replaces tradition to have serum free culture system liquid with mentioned component, has avoided the impact of serum component on experimental study.Compared with the conventional method, serum-free medium of the present invention can suppress fibroblastic growth in primary cell culture process effectively, thereby can obtain the epithelial cell that purity is higher.
Accompanying drawing explanation
Figure 1 shows that according to the primary submaxillary duct epithelial cell of the serum-free cell culture medium cultivator figure of the embodiment of the present invention 2 preparations.
Figure 2 shows that according to the primary submaxillary gland acinus of the serum-free cell culture medium cultivator epithelial cell figure of the embodiment of the present invention 2 preparations.
Figure 3 shows that according to the primary parotid gland epithelial cell of the serum-free cell culture medium cultivator figure of the embodiment of the present invention 2 preparations.
Figure 4 shows that according to the primary sublingual gland epithelial cell of the serum-free cell culture medium cultivator figure of the embodiment of the present invention 2 preparations.
Figure 5 shows that according to the serum-free cell culture medium of the embodiment of the present invention 2 preparations and cultivate rat submandibular gland epithelial cell figure.
Figure 6 shows that according to the serum-free cell culture medium of the embodiment of the present invention 2 preparations and cultivate mouse primary pancreas epithelial cell figure.
Figure 7 shows that according to the serum-free cell culture medium of the embodiment of the present invention 2 preparations and cultivate mouse primary epithelial cells of submandibular gland figure.
Figure 8 shows that according to the serum-free cell culture medium of the embodiment of the present invention 2 preparations and cultivate mouse submandibular gland epithelial cell line the 42nd generation cytological map.
Figure 9 shows that the present invention does not scheme containing the serum-free cell culture medium cultivator submaxillary duct stem cell of BPE.
Figure 10 (a)-10 (d) is depicted as serum-free cell culture medium 3 D stereo cultivator submaxillary duct stem cell figure prepared in accordance with the present invention.
Figure 11 shows that the karyotyping of cultivating mouse submandibular gland epithelial cell line the 42nd generation cell according to the serum-free cell culture medium of the embodiment of the present invention 2 preparations.
Figure 12 (a) and 12 (b) are depicted as the present invention and containing the serum-free cell culture medium of BPE, do not cultivate the reactivity of the 42nd generation of mouse submandibular gland epithelial cell line to epithelical cell growth factor EGF and fibroblast growth factor FGF1.
Figure 13 shows that the present invention does not cultivate the reactivity of mouse submandibular gland epithelial cell to epithelical cell growth factor EGF, fibroblast growth factor FGF1 and Medulla Bovis seu Bubali extract BPE containing the serum-free cell culture medium of BPE.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention, should be understood that these embodiment are only not used in restriction range of application of the present invention for the present invention is described.
The serum-free medium that the present invention cultivates for Mammals sialisterium epithelial cell, in basic medium MCDB153HAA, to add following composition: amino acid, VITAMIN, salt, lipid, trace element, hormone, buffer reagent and glucose, nonessential amino acid, thymidine, xanthoglobulin, phenol red, beta-mercaptoethanol, Transferrins,iron complexes, bovine serum albumin, the content of its each component is shown in shown in following embodiment 1～6, and by components dissolved in each embodiment, in without thermal source ultrapure water, (resistivity is 18.2 Ω) configuration forms.
Embodiment 1
Embodiment 2
Embodiment 3
Embodiment 4
Compare with embodiment 1, in this embodiment, do not add BPE, add in addition L-AA-2-tricresyl phosphate sodium salt of 9.5mg/L, the heparin sodium of 0.3mg/L, the NSC 334200 dipeptides of the FGF2 of 5 μ g/L and 400mg/L, other added ingredientss and content are identical with embodiment 1.
Embodiment 5
Compare with embodiment 2, in this embodiment, do not add BPE, add in addition L-AA-2-tricresyl phosphate sodium salt of 11.5mg/L, the heparin sodium of 0.6mg/L, the NSC 334200 dipeptides of the FGF2 of 15 μ g/L and 420mg/L, other added ingredientss and content are identical with embodiment 2.
Embodiment 6
Compare with embodiment 3, in this embodiment, do not add BPE, add in addition L-AA-2-tricresyl phosphate sodium salt of 12.5mg/L, the heparin sodium of 1mg/L, the NSC 334200 dipeptides of the FGF2 of 20 μ g/L and 460mg/L, other added ingredientss and content are identical with embodiment 3.
Embodiment 7
As shown in Figure 9, the serum-free cell culture medium cultivator submaxillary duct epithelial stem cell figure for preparing according to the embodiment of the present invention 5.In this embodiment, for cultivating the sialisterium epithelial stem cell obtaining for 5-7 days.
Embodiment 8
The method of applying three-dimensional stereoscopic culture, observes conduit formational situation.Cultivate after 10 days, utilize the analytical procedure of immunofluorescence dyeing, the expression of α-amylase in the sialisterium stem cell of detection embodiment 7.As shown in Figure 10 (a)-10 (d), in Matrigel, stem cell forms catheter-like, and blue-fluorescence is nucleus dyestuff DAPI, and green fluorescence is α-amylase (sialisterium epithelial cell specificity molecular marker) antibody staining.
Embodiment 9
Utilize method of karyotype analysis, detect mouse submandibular gland epithelial cell line.As shown in figure 11, be the karyotyping that serum-free cell culture medium of the present invention is cultivated mouse submandibular gland epithelial cell line the 42nd generation cell, result shows that caryogram is normal, is diploid, shows after long-term cultivation and continuous passage the state that cell is still kept fit.
Embodiment 10
Figure 12 shows that the present invention does not cultivate the reactivity of the 42nd generation of mouse submandibular gland epithelial cell line to epithelical cell growth factor EGF and fibroblast growth factor FGF1 containing the serum-free cell culture medium of BPE.Cell in logarithmic phase is sowed to 24 orifice plates to 10000, every hole cell; The EGF or the FGF1 that after 24 hours, add respectively different concns.As Figure 12 (a) with (b), somatomedin is added 5 days countings afterwards.Result shows, the 42nd generation cell still two kinds of somatomedins are had to significant reaction, with the 7th generation cell there is no notable difference.Circular mark represent the 7th generation cell, triangular marker represent the 42nd generation cell.
Figure 13 shows that the present invention does not cultivate the reactivity of mouse submandibular gland epithelial cell to epithelical cell growth factor EGF, fibroblast growth factor FGF1 and Medulla Bovis seu Bubali extract BPE containing the serum-free cell culture medium of BPE.EGF (1nM) and FGF (100pM) are added in the circular mark representative of white simultaneously, FGF (100pM) is only added in the representative of square mark, EGF (1nM) is only added in triangular marker representative, and BPE (15mg/mL) is added in the circular mark representative of black.As shown in figure 13, a certain amount of somatomedin is calculated cell quantity every day after adding, and amounts to 6 days.Result shows, adds BPE and can significantly promote cell proliferation in nutrient solution.
Claims (2)
1. the serum-free medium of cultivating for Mammals sialisterium epithelial cell, it is characterized in that, be in basic medium, to add amino acid, VITAMIN, salt, lipid, trace element, damping fluid, hormones compound, turn metalloprotein, antioxidant, serum albumin, carbohydrate, purine, pyrimidine bases material and pH pH indicator;
Wherein, described basic medium is MCDB153HAA substratum; The component and the addition that add are as follows
In described nutrient solution, also add 12～15mg/L Medulla Bovis seu Bubali extract.
2. a compound method for serum-free medium as claimed in claim 1, is characterized in that comprising the following steps
(1) take MCDB153HAA as basic medium, add amino acid, VITAMIN, salt, lipid, trace element, damping fluid, hormones compound, turn metalloprotein, antioxidant, serum albumin, carbohydrate, purine, pyrimidine bases material and pH pH indicator;
(2) add the Medulla Bovis seu Bubali extract of 12～15mg/L in the resulting nutrient solution of step (1).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110303107.XA CN102399742B (en) | 2011-09-30 | 2011-09-30 | Serum-free culture fluid for culture of salivary gland epidermal cell and salivary gland stem cell of mammals |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110303107.XA CN102399742B (en) | 2011-09-30 | 2011-09-30 | Serum-free culture fluid for culture of salivary gland epidermal cell and salivary gland stem cell of mammals |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102399742A CN102399742A (en) | 2012-04-04 |
| CN102399742B true CN102399742B (en) | 2014-07-30 |
Family
ID=45882394
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110303107.XA Active CN102399742B (en) | 2011-09-30 | 2011-09-30 | Serum-free culture fluid for culture of salivary gland epidermal cell and salivary gland stem cell of mammals |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102399742B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102766662A (en) * | 2012-07-30 | 2012-11-07 | 中粮生物化学（安徽）股份有限公司 | Culture medium and application of culture medium and lysine flask shaking fermentation method |
| US9321995B2 (en) | 2012-12-20 | 2016-04-26 | Suzhou Biowisetech Co., Ltd. | Stem cell culture medium and its applications as well as a stem cell culture method |
| CN103060264B (en) * | 2012-12-20 | 2015-01-21 | 上海市第十人民医院 | Stem cell culture medium and application thereof and stem cell cultivation method |
| CN105543174A (en) * | 2016-03-01 | 2016-05-04 | 赛百慷（上海）生物技术股份有限公司 | Tumor cell growth promoting additive and application method thereof |
| CN105647850A (en) * | 2016-03-01 | 2016-06-08 | 赛百慷（上海）生物技术股份有限公司 | Epithelium culture system |
| CN106591219A (en) * | 2016-12-22 | 2017-04-26 | 叶宗耀 | Culture medium for epidermal cells |
| CN106811440B (en) * | 2017-02-09 | 2020-09-15 | 广西壮族自治区水牛研究所 | Method for separating and culturing buffalo mammary epithelial cells from buffalo milk |
| CN109749997B (en) * | 2018-05-11 | 2020-03-17 | 中山大学中山眼科中心 | Limbal stem cell serum-free medium and culture method thereof |
| JPWO2020203850A1 (en) * | 2019-03-29 | 2020-10-08 | ||
| US20220290097A1 (en) * | 2019-08-03 | 2022-09-15 | Shenzhen Mytang Biotechnology Co., Ltd. | Poultry Egg-Based Culture Medium |
| WO2022130162A1 (en) * | 2020-12-14 | 2022-06-23 | Clear Meat Private Limited | A supplement composition for cell culture |
| CN113712195B (en) * | 2021-08-06 | 2023-08-08 | 澳门大学 | Artificial bird's nest and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997034999A1 (en) * | 1996-03-18 | 1997-09-25 | University Of Pittsburgh | Cell culture media for mammalian cells |
| CN101044235A (en) * | 2004-09-08 | 2007-09-26 | 威斯康星校友研究基金会 | Culturing human embryonic stem cells |
| CN101984051A (en) * | 2010-11-19 | 2011-03-09 | 中山大学 | Serum-free cell culture fluid suitable for enriching and culturing tumour stem cells |
-
2011
- 2011-09-30 CN CN201110303107.XA patent/CN102399742B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997034999A1 (en) * | 1996-03-18 | 1997-09-25 | University Of Pittsburgh | Cell culture media for mammalian cells |
| CN101044235A (en) * | 2004-09-08 | 2007-09-26 | 威斯康星校友研究基金会 | Culturing human embryonic stem cells |
| CN101984051A (en) * | 2010-11-19 | 2011-03-09 | 中山大学 | Serum-free cell culture fluid suitable for enriching and culturing tumour stem cells |
Non-Patent Citations (2)
| Title |
|---|
| Isoleucine prevents rat salivary gland epithelial cells from apoptosis in serum-free culture;Miho Furue et al;《In vitro cellular and developmental biology animal》;20000531;第36卷(第5期);第287页左栏第1段、右栏第1段，第288页左栏第1段 * |
| Miho Furue et al.Isoleucine prevents rat salivary gland epithelial cells from apoptosis in serum-free culture.《In vitro cellular and developmental biology animal》.2000,第36卷(第5期), |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102399742A (en) | 2012-04-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102399742B (en) | Serum-free culture fluid for culture of salivary gland epidermal cell and salivary gland stem cell of mammals | |
| CN103060264B (en) | Stem cell culture medium and application thereof and stem cell cultivation method | |
| CN101760442B (en) | Serum-free medium for MDCK cell large-scale adherent culture and single-cell suspension culture | |
| ES2660898T3 (en) | Method for preparing a basic culture medium for mesenchymal stem cells, basic culture medium for mesenchymal stem cells, and cultured and differentiated cellular therapeutic agent using the same | |
| GB2433943A (en) | Cultivation of primate embryonic stem cells | |
| CN104818248A (en) | Immunocyte culture medium, and culture method and application of immunocytes | |
| CN107043747A (en) | A kind of human umbilical cord mesenchymal stem cells serum free medium | |
| CN105886464A (en) | Serum-free culture medium for umbilical cord blood mesenchymal stem cells | |
| CN102634482A (en) | Serum-free complete medium for mesenchymal stem cell | |
| CN103468641B (en) | Efficient amplification method of TILs serving as sources of cancerous pleural effusion | |
| CN104911143A (en) | Protein-free, hydrolysate-free and serum-free culture medium and preparation method thereof | |
| CN110004114A (en) | A kind of serum free medium of dental pulp stem cell | |
| CN101418330A (en) | Non protein culture medium adapted to large-scale culture of NSO cell and production of antibody | |
| CN106834229A (en) | For the serum free medium of people's immunologic cytotoxicity cell expansion ex vivo | |
| CN105462912A (en) | Protein-free serum-free medium suitable for diploid cell culture and application | |
| CN102719393B (en) | Serum-free medium capable of inducing tumor stem cells for differentiation towards lymphatic endothelial cells, and method therefor | |
| CN104357379A (en) | Stem cell culture medium | |
| CN105838675A (en) | Hematopoietic stem cell serum-free culture medium | |
| CN107236708A (en) | A kind of serum free medium for supporting HeLa cell attachment cultures | |
| CN105524882A (en) | Serum substitution combination for immune killer cell amplification in vitro | |
| CN106754634A (en) | A kind of serum free medium and preparation method thereof | |
| CN106148269A (en) | A kind of mammalian cell culture and additive thereof | |
| CN104164404A (en) | Application of serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro | |
| EP3858980A1 (en) | Culture additive, culture medium and culture method for animal cells | |
| CN106754677A (en) | A kind of external mesenchymal stem cells MSCs culture medium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |