CN105368769A - Method for preparation of cell culture active component composition and cell culture medium - Google Patents
Method for preparation of cell culture active component composition and cell culture medium Download PDFInfo
- Publication number
- CN105368769A CN105368769A CN201510794654.0A CN201510794654A CN105368769A CN 105368769 A CN105368769 A CN 105368769A CN 201510794654 A CN201510794654 A CN 201510794654A CN 105368769 A CN105368769 A CN 105368769A
- Authority
- CN
- China
- Prior art keywords
- cell
- active ingredient
- conditioned medium
- cell culture
- medium liquid
- 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.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention discloses a method for preparation of a cell culture active component composition and a cell culture medium. The method for preparation of the cell culture active component composition comprises the following steps: a first cell, which is a mammalian adipose stem cell, is cultured until the fusion rate is more than 80%, and then the first cell is removed to obtain a first cell supernatant; a second cell, which is mammalian fibroblast, is cultured until the fusion rate is more than 80%, and then the second cell is removed to obtain a second cell supernatant; and the first cell supernatant and the second cell supernatant are mixed to obtain the cell culture active component composition. The cell culture medium comprises a cell basic nutrient medium and the cell culture active component composition prepared by the method.
Description
Technical field
The present invention relates to field of cell culture, be specifically related to the active ingredient compositions of cell cultures and the preparation of relevant substratum and use.
Background technology
Stem cell is the special cell of a class, and it has the of self-replication capacity, and under given conditions, can be divided into several functions cell.According to different sorting techniques, stem cell can be divided into embryonic stem cell, adult stem cell, or myeloid-lymphoid stem cell, multipotential stem cell, specially energy stem cell etc.Stem cell has tempting prospect in reparation and the regeneration of damaged tissue organ, efficient medicating active ingredients are prepared etc.Research and development based on stem cell culture technique were sharply increasing in recent years, became new medicine and the study hotspot of health field.
The perfect medium of culturing cell is made up of basic medium (as MEM and DMEM etc.) and additive (hormone and somatomedin as serum or serum-free culture).Therefore, according to whether containing serum, cell culture medium can be divided into two classes, the substratum namely containing serum and serum free medium.Serum contains the breeding that somatomedin can promote cell, can promote the adherent of cell containing attachment element, also has antisteapsin activity simultaneously.Serum is also the source of mineral substance, lipid and hormone simultaneously.But, a major defect of the substratum containing serum has animal derived components, some composition is not also studied clear completely, in the experiment relevant with drug research, animal derived components may bring the problem of Biosafety, from this angle, when cell cultures, serum content is more few better.
Serum free medium generally forms by nutritive ingredient with bioactive molecules, generally need interpolation tens kinds to tens kinds bioactive molecules, relative populations proportionlity between each bioactive molecule is difficult to adjust to optimum regime, thus is difficult to the optimal growth performance playing cell.
In stem cell culturing process, cell culture medium is the key factor of stem cell growth differentiation.Due to the singularity of stem cell, especially high to the requirement of substratum.Existing stem cell serum-free culture medium is made up of nutritional type additive and bioactive molecule, but the ratio of each bioactive molecule is difficult to accurate assurance, culturing stem cells particularly early stage stem cell time be difficult to the maximum growth ability playing cell.In addition when treating some disease by stem cell culture, particularly when disease relates to multiple bioactive molecules, culture prepared by traditional stem cell media is difficult to prove effective.
A kind of serum free medium cultivating placenta mesenchyma stem cell disclosed in Chinese patent application CN103805562B, based on DMEM nutrient solution, also containing fibroblast growth factor acceptor 2, tethelin, Regular Insulin, Transferrins,iron complexes, gsh, BMP-4, L-glutaminate, Sodium.alpha.-ketopropionate, non-essential amino acid and beta-mercaptoethanol etc.
The and for example fat stem cell substratum of a kind of serum-free disclosed in Chinese patent application CN104877962A, comprise IMDM basic medium, also comprise following component: recombinant human insulin, recombinant transferrin, vitamins C, human serum albumin, fibroblast growth factor, Thr6 PDGF BB, Urogastron, rhIGF-1, transforming growth factor, fibronectin, Pp63 glycophosphoproteins, tea-polyphenol and stem cell factor.
In sum, this area still needs to be suitable for the cell cultures especially activeconstituents cultivated of stem cell and substratum.
Summary of the invention
For solving the problems of the technologies described above, the invention provides a kind of novel cell cultures active ingredient compositions that can meet Growth of Cells demand and preparation method thereof and the substratum comprising this active ingredient compositions and uses thereof.
Active ingredient compositions of the present invention and the cell culture medium containing said composition overcome the existing shortcoming containing blood serum medium and serum free medium.As described in previous Background, existing cell culture medium is divided into two kinds, contains or does not contain serum.The major defect of the substratum containing serum has animal derived components, and in the experiment relevant with drug research, animal derived components may bring the problem of Biosafety.On the other hand, serum free medium is generally made up of nutritional type additive and tens kinds to tens kinds bioactive molecules, and the ratio of each bioactive molecule is difficult to accurate assurance, thus is difficult to the maximum growth ability playing cell.
According to a first aspect of the invention, a kind of method preparing cell cultures active ingredient compositions is provided, comprises the following steps: 1) cultivate the first cell, cell is mammal fat stem cell, be cultured to fusion rate more than 80%, removing cell, obtains the first cell conditioned medium liquid; 2) cultivate the second cell, cell is mammalian fibroblasts, is cultured to fusion rate more than 80%, removing cell, obtains the second cell conditioned medium liquid; And 3) by above two kinds of supernatant liquors mixing, thus make cell cultures active ingredient compositions.
In a preferred implementation, the volume ratio of the first cell conditioned medium liquid and the second cell conditioned medium liquid is 1:0.2 ~ 5.
In another preferred implementation, the method also comprises the step of cultivating the third cell, the third cell and stand-by cell cultures active ingredient compositions cultured cells come from same species, be cultured to fusion rate more than 85%, removing cell, obtain the third cell conditioned medium liquid, and the three kinds of supernatant liquor mixing that will obtain, thus make cell cultures active ingredient compositions.
More preferably, the volume ratio of the first cell conditioned medium liquid and the second cell conditioned medium liquid is 1:0.2 ~ 5, and the volume ratio of the first cell conditioned medium liquid and the third cell conditioned medium liquid is 1:0.2 ~ 5.
Selectively, the first cell, the second cell and the third cell come from same Mammals respectively, such as the mankind, apes or muroid.
Selectively, the first cell, the second cell and the third cell can come from different Mammalss respectively, such as: the first cell and the second cell come from the mankind, and the third cell comes from muroid.
In another preferred implementation, also comprise and add the step that at least one is selected from following activeconstituents in supernatant liquor mixed solution: Regular Insulin, Pp63 glycophosphoproteins, linolic acid, Transferrins,iron complexes, dexamethasone, fibroblast growth factor or Sodium Selenite.
Preferably, add Regular Insulin, Pp63 glycophosphoproteins, linolic acid, Transferrins,iron complexes, dexamethasone, fibroblast growth factor, Sodium Selenite respectively in supernatant liquor mixed solution, make the final concentration of above composition be respectively 0.2-17.2ng/L, 0.2 ~ 0.8mg/mL, 0.5 ~ 2 μ g/mL, 30 ~ 70 μ g/mL, 0.025-2.54ng/L, 80 ~ 150ng/mL, 0.12-0.29ng/L.
Second aspect present invention provides the cell cultures prepared with aforesaid method of the present invention active ingredient compositions.
Third aspect present invention provides a kind of cell culture medium, comprises cell base nutritional medium and active ingredient compositions of the present invention.
Preferably, the volume ratio of cell cultures active ingredient compositions and cell base nutritional medium is 1:5 ~ 20.
Wherein, cell base nutritional medium includes but not limited to DMEM or MEM substratum.
Fourth aspect present invention provides a kind of cell culture processes, and described method comprises the step using active ingredient compositions of the present invention or cell culture medium of the present invention.In a preferred implementation, treat that cultured cells is stem cell, preferred fat stem cell or mesenchymal stem cells MSCs.
Fifth aspect present invention provides active ingredient compositions of the present invention or the purposes of cell culture medium of the present invention in cell cultures.In a preferred implementation, treat that cultured cells is stem cell, preferred fat stem cell or mesenchymal stem cells MSCs.
The invention has the beneficial effects as follows: compared with the substratum of prior art, active ingredient compositions of the present invention comprises specific cell culture supernatant, cell culture medium containing inventive compound composition not containing serum (namely not containing other animal derived components), and adds other less bioactive molecules.Containing the various active composition required for Growth of Cells in active ingredient compositions of the present invention, and these activeconstituentss ratio is each other the ratio under native state, meets the natural demand of Growth of Cells, thus can ensure the optimum growh of cell.In addition, when the culture that when needing to relate to multiple bioactive molecules by the disease of stem-cell therapy prepared by traditional stem cell media is difficult to prove effective, the stem cell culture of culture medium culturing of the present invention is utilized can to reach good result for the treatment of.
Embodiment
Elaborate further below by making the present invention with reference to embodiment, but these elaborations do not limit in any form the present invention.Unless otherwise stated, the implication that all Science and Technology terms used herein have belonging to the present invention and the those skilled in the art of correlative technology field understand usually.
The invention provides a kind of method preparing cell cultures active ingredient compositions, said method comprising the steps of: 1) cultivate the first cell, described cell is mammal fat stem cell, is cultured to fusion rate more than 80%, removing cell, obtains the first cell conditioned medium liquid; 2) cultivate the second cell, described cell is mammalian fibroblasts, is cultured to fusion rate more than 80%, removing cell, obtains the second cell conditioned medium liquid; And 3) by above two kinds of supernatant liquors mixing, thus make described cell cultures active ingredient compositions.
In a preferred implementation, the first cell conditioned medium liquid described: the volume ratio of the second cell conditioned medium liquid is 10:1 to 1:10, preferred 5:1 to 1:5, most preferably 1:1.In another preferred implementation, fusion rate is 80%-97%, preferred 85%-95%.Filtration or the mode known to other those skilled in the art can be adopted from cell culture fluid, to remove cell to obtain supernatant liquor.Step 1 in aforesaid method) and step 2) in no particular order sequentially.
In another preferred implementation, aforesaid method is cultivated except the first and the second cell except comprising, also optionally comprise and cultivate the third cell, especially when expect with cell culture medium cultured cells of the present invention and described the first and/or the second cell does not derive from same species time, the third cell of preferred cultivation, this third cell comes from same species with expection by cell culture medium cultured cells of the present invention.Such as, if for the preparation of the substratum of mouse embryonic fibroblast, then select other cells of rat hepatocytes or mouse as the third cell.By the third cell cultures to fusion rate more than 85%, removing cell, obtains the third cell conditioned medium liquid, and the three kinds of supernatant liquor mixing that will obtain, thus make described cell cultures composition.The third cell conditioned medium liquid and the volume ratio between the first and the mixed solution of the second cell conditioned medium liquid are 9:1 to 1:9, preferred 5:1 to 1:5, most preferably 1:1.In a preferred implementation, by the third cell cultures to fusion rate 85%-97%.In another preferred implementation, by the third cell cultures such as, to fusion rate more than 90%, 90%-97%, preferred 90%-95%.The third cell preferred mammal cell.The third cell can be the liver cell of liver cell, such as people or mouse.
The cultivation of three kinds of cells is order in no particular order, and after cell cultures, can collect supernatant liquor immediately to use or by frozen for supernatant liquor stand-by.The cultivation of three kinds of cells, such as can be see all according to any suitable method known to those skilled in the art, " EssentialsofStemCellBiology ", RobertLanza etc., (2006); The simple and easy method of rat hepatocytes separation and original cuiture, Liu Xuezhong etc., Jiangsu's agriculture journal, 2009,25 (1): 222 ~ 224; The progress of fat mesenchymal stem cell, yellow scorching Na etc., HEILONGJIANG ANIMAL SCIENCE AND VETERINARY MEDICINE science and technology version, 2011,7:46-48; The original cuiture of rat hepatocytes and qualification, Liu Demin etc., Tianjin medicine, 2006,34 (5): 322-323.
Mammals described in the present invention includes but not limited to people, ape, rhesus monkey, hamster, rabbit, horse, pig, rat, mouse etc.Preferred people, rat or mouse.
Another preferred embodiment in, as required, method of the present invention also comprises adds the step that one or more are selected from following activeconstituents: Regular Insulin, Pp63 glycophosphoproteins, linolic acid, Transferrins,iron complexes, dexamethasone, FGF (fibroblast growth factor) or Sodium Selenite, to adapt to the needs of different Growth of Cells and/or differentiation, if added when such as dexamethasone is mainly used in the demand of Cell differentiation inducing activity; FGF is somatomedin, plays FGF deficiency in supplementary nutrient solution; Other are added ingredientss of nutritional type.One preferred embodiment in, based on final cell culture medium, the concentration that above-mentioned activeconstituents adds can be: Regular Insulin 10
-5-10
-7m, Pp63 glycophosphoproteins 0.2-0.8mg/mL, linolic acid 0.5-2 μ g/mL, Transferrins,iron complexes 25-75 μ g/mL, dexamethasone 10
-6-10
-8m, FGF50-200ng/mL, Sodium Selenite 3 × 10
-7-3 × 10
-9m; Most preferably, Regular Insulin 10 is added
-6m, Pp63 glycophosphoproteins 0.5mg/mL, linolic acid 1 μ g/mL, Transferrins,iron complexes 51 μ g/mL, dexamethasone 10
-7m, FGF100ng/mL, and Sodium Selenite 3 × 10
-8m.
One preferred embodiment in, active ingredient compositions of the present invention or cell culture medium of the present invention are used for stem cell, the fat stem cell (fat mesenchymal stem cell of such as people or mouse) of such as fat stem cell (such as, fat mesenchymal stem cell), especially people or mouse or the cultivation of mesenchymal stem cells MSCs (such as rat bone marrow mesenchymal stem cells).
For object of the present invention, term " fat stem cell (adipose-derivedstemcells; ADSCs) " refers to the stem cell deriving from fatty tissue, and specifically, fat stem cell is from fatty tissue, be separated the stem cell with multi-lineage potential obtained.In the present invention, fatty tissue or fatty raw material are not particularly limited, and can be the fatty tissues at any position deriving from animal or human, the fatty tissue of preferred mammal especially people.
Term " inoblast " herein comprises the inoblast in all sources, such as dermal fibroblast (such as fibroblasts of adult human dermis), myofibroblast etc.
Herein, term " cell conditioned medium liquid ", " cell culture supernatant " and " supernatant liquor " are used interchangeably, phalangeal cell after cultivating, the liquid obtained after removing cell such as filtering cell.
The present invention also provides the cell cultures prepared with aforesaid method of the present invention active ingredient compositions.Said composition is the mixed solution of two or three cell culture supernatant.The method preparation of described composition by comprising the following steps: 1) cultivate the first cell, described cell is mammal fat stem cell, is cultured to fusion rate more than 80%, and removing cell, obtains the first cell conditioned medium liquid; 2) cultivate the second cell, described cell is mammalian fibroblasts, is cultured to fusion rate more than 80%, removing cell, obtains the second cell conditioned medium liquid; And 3) by above two kinds of supernatant liquors mixing, thus make described cell cultures active ingredient compositions.
In a preferred implementation, aforesaid method is cultivated except the first and the second cell except comprising, also optionally comprise and cultivate the third cell, especially when expect with cell culture medium cultured cells of the present invention and described the first and/or the second cell does not derive from same species time, the third cell of preferred cultivation, this third cell comes from same species with expection by cell culture medium cultured cells of the present invention.Such as, if for the preparation of the substratum of mouse embryonic fibroblast, then select other cells of rat hepatocytes or mouse as the third cell.By the third cell cultures to fusion rate more than 85%, removing cell, obtains the third cell conditioned medium liquid, and the three kinds of supernatant liquor mixing that will obtain, thus make described cell cultures composition.
The present invention also provides a kind of cell culture medium, comprises cell base nutritional medium and active ingredient compositions of the present invention, makes by being added in cell base nutritional medium by active ingredient compositions of the present invention.The amount that active ingredient compositions of the present invention adds in basal nutrient substratum is, active ingredient compositions accounts for the 5-25 volume % of basal nutrient substratum, preferred 10-20 volume %.
" basal nutrient substratum " or " cell base nutritional medium " in the present invention is used interchangeably, and all gets its broad sense, and namely referring to can provide any suitable substratum of basal nutrient demand for the cell of required type.Basal nutrient substratum includes but not limited to DMEM (Dulbecco'smodifiedEagle'smedium) or MEM (minimumessentialmedium) substratum.
Also can according to specific needs (such as stem cell directional is induced to differentiate into concrete cell type) and add other compositions in cell culture medium of the present invention.
The present invention also provides a kind of cell culture processes, and described method comprises the step using active ingredient compositions of the present invention or cell culture medium of the present invention.
The present invention provides active ingredient compositions of the present invention or the purposes of cell culture medium of the present invention in cell cultures in addition, described cell cultures is cultivated by preferred stem cell, such as fat stem cell (such as, fat mesenchymal stem cell), especially human adipose-derived stem cell (such as human adipose mesenchymal stem cells), or mesenchymal stem cells MSCs (such as rat bone marrow mesenchymal stem cells).
The present invention is set forth further below in conjunction with specific embodiment.Should be understood that the scope that these embodiments only do not limit the present invention in any way for illustration of the present invention.Scope of the present invention is limited only by the accompanying claims.Those skilled in the art are after having read present disclosure, can make multiple change and change to the present invention without departing from the spirit and scope of the present invention, these are changed and change all should be thought the equivalents of embodiment of the present invention and fall within the scope of the invention.In addition, each feature of the present invention (comprising above specification sheets and embodiment hereafter and claims) in the application's book and set forth can combine mutually and form new feature, as space is limited, repeat no more herein, but it may be noted that these new features are also in the scope that the present invention is contained.
Embodiment
Embodiment 1
One. the preparation of human adipose-derived stem cell culture supernatant
1. the preparation of human adipose-derived stem cell substratum
Use the MesenPRO of Gibco company to subtract substratum based on serum cell culture medium (serum content 2%), be stored in 4 DEG C in dark place after thawing, and be finished in 15 days.
2. cell prepares
Human adipose-derived stem cell, purchased from Chinese Academy of Sciences's Shanghai cell bank, shifts in 50ml centrifuge tube, centrifugal about 3 minutes of 1500rpm, with MesenPRO substratum, precipitation is blown afloat, get 100 μ L, 0.4% Trypan Blue microscopy, situation in the record visual field, and count with blood counting chamber.
3. inoculate
Human adipose-derived stem cell is seeded to healthy and free from worry 225 ml cells culturing bottles, and every bottle of inoculum size is 2 × 10
6individual cell.Jog is to make cell dispersal even.
4. human adipose-derived stem cell culturing bottle is carried out cultivating (carbon dioxide content 5%) in 37 DEG C of incubators, reach more than 85% to cell confluency.
5. jog culturing bottle, is then transferred to the filter membrane of human adipose-derived stem cell culture supernatant by one 0.2 μm in aseptic polycarbonate bottles.
6. human adipose-derived stem cell passage, goes down to posterity once in every 7 days, ensures that every cell bottle (225 milliliters) cell quantity is not less than 2 × 10 after going down to posterity at every turn
6individual.
7. as step 5 method is collected frozen in-20 DEG C at every turn before the human adipose-derived stem cell culture supernatant after going down to posterity went down to posterity next time.
Two. the preparation of human fibroblasts's culture supernatant
8. the preparation of human fibroblasts
Human fibroblasts is purchased from Chinese Academy of Sciences's Shanghai cell bank, shift in 50ml centrifuge tube, centrifugal about 3 minutes of 1500rpm, cell precipitation ScienCell company fibroblast culture medium (article No. 2301) is blown afloat, get 100 μ L, 0.4% Trypan Blue microscopy, situation in the record visual field, and count with blood counting chamber.
9. inoculate
Human fibroblasts is seeded to healthy and free from worry 225 ml cells culturing bottles, and every bottle of inoculum size is 2 × 10
6individual cell.Jog is to make cell dispersal even.
10. culturing bottle is carried out cultivating (carbon dioxide content 5%) in 37 DEG C of incubators, reach more than 85% to cell confluency.
11. jog culturing bottles, are then transferred to the filter membrane of human fibroblasts's culture supernatant by one 0.2 μm in aseptic polycarbonate bottles.
12. human fibroblasts's passages, go down to posterity once in every 4 days, ensure that every cell bottle (225 milliliters) cell quantity is not less than 2 × 10 after going down to posterity at every turn
6individual.
13. go down to posterity at every turn after human fibroblasts's culture supernatant go down to posterity next time before as step 11 method collect frozen in-20 DEG C.
Three. mixing
The 14. frozen supernatant liquors getting above first part, second section, after melting respectively, get equivalent volumes (volume ratio 1:1), and slight oscillatory mixing on shaking table, mixed solution is for subsequent use.
Four. prepare substratum and culturing cell
15. by the mixed solution in step 14 in 10% ratio be added into DMEM substratum, be adipose-derived stem cells special culture media.
The 16. culture medium culturing human adipose mesenchymal stem cells using aforesaid method to prepare, 37 DEG C, CO2gas incubator (gas concentration lwevel 5%), cultivates cell after 2 days and starts adherent, within 5 days, can be paved with one deck.
Embodiment 2
One. prepared by mouse adipocytes culture supernatant
1. the preparation of mouse adipocytes substratum
Use the M199 cell culture medium (increase serum content 5%) of Hyclone company, mouse adipocytes is purchased from Chinese Academy of Sciences's Shanghai cell bank.
2. inoculate
Mouse adipocytes is seeded to healthy and free from worry 225 ml cells culturing bottles, and every bottle of inoculum size is 2 × 10
6individual cell.Jog is to make cell dispersal even.
3. mouse adipocytes culturing bottle is carried out cultivating (carbon dioxide content 5%) in 37 DEG C of incubators, reach more than 85% to cell confluency.
4. jog culturing bottle, is then transferred to the filter membrane of mouse adipocytes culture supernatant by one 0.2 μm in aseptic polycarbonate bottles.
5. mouse adipocytes passage, goes down to posterity once in every 4 days, ensures that every cell bottle (225 milliliters) cell quantity is not less than 2 × 10 after going down to posterity at every turn
6individual.
6. as step 4 method is collected frozen in-20 DEG C at every turn before the mouse adipocytes culture supernatant after going down to posterity went down to posterity next time.
Two. the preparation of mouse muscle-forming cell culture supernatant
7. the preparation of mouse muscle-forming cell substratum
Mouse muscle-forming cell is purchased from Chinese Academy of Sciences's Shanghai cell bank, shift into containing in 50ml centrifuge tube, centrifugal about 3 minutes of 1500rpm, cell precipitation ScienCell company fibroblast culture medium (article No. 2301) is blown afloat, get 100 μ L, 0.4% Trypan Blue microscopy, situation in the record visual field, and count with blood counting chamber.
8. inoculate
Mouse muscle-forming cell is seeded to healthy and free from worry 225 ml cells culturing bottles, and every bottle of inoculum size is 2 × 10
6individual cell.Jog is to make cell dispersal even.
9. culturing bottle is carried out cultivating (carbon dioxide content 5%) in 37 DEG C of incubators, reach more than 85% to cell confluency.
10. jog culturing bottle, is then transferred to the filter membrane of mouse muscle-forming cell culture supernatant by one 0.2 μm in aseptic polycarbonate bottles.
11. mouse muscle-forming cell passages, go down to posterity once in every 4 days, ensure that every cell bottle (225 milliliters) cell quantity is not less than 2 × 10 after going down to posterity at every turn
6individual.
12. go down to posterity at every turn after mouse muscle-forming cell culture supernatant go down to posterity next time before as step 5 method collect frozen in-20 DEG C.
Three. prepared by mice embryonic liver cell culture supernatant liquor
13. mice embryonic liver cells are purchased from Chinese Academy of Sciences's Shanghai cell bank, and substratum uses the HyQ-RS substratum of Hytclone, and cultural method, with step 7-12, collects supernatant liquor, frozen for subsequent use.
Four. mixing
14. get above frozen supernatant liquor, after melting respectively, in mouse adipocytes: mouse muscle-forming cell: mice embryonic liver cell ratio is 4:4:2 (volume ratio) mixing.
Four. add other active substances
15. respectively to adding Regular Insulin, Pp63 glycophosphoproteins, linolic acid, Transferrins,iron complexes, dexamethasone, fibroblast growth factor (FGF), Sodium Selenite in mixed solution, final concentration is made to be respectively 10ng/L, 0.5mg/mL, 1 μ g/mL, 51 μ g/mL, 1ng/L, 100ng/mL, 0.2ng/L, then filtered respectively by 0.2 μm of filter membrane, mix for subsequent use with the filtrate of step 14.
16. by the mixed solution in step 15 in 10% ratio be added into MEM substratum, be mouse embryo fibroblasts special culture media.
The 17. culture medium culturing mouse embryo fibroblasts using aforesaid method to prepare, 37 DEG C, CO2gas incubator (gas concentration lwevel 10%), cultivates cell attachment after 5 days, within 8-9 days, can be paved with one deck.
Embodiment 3
One. first two cell culture processes and supernatant liquor preparation are with embodiment 1.
Two. the preparation of rat hepatocytes culture supernatant
1. rat hepatocytes is purchased from Chinese Academy of Sciences's Shanghai cell bank, and substratum uses GIBCO, article No. 12800017 substratum, and 5% CO2gas incubator is cultivated, and temperature 37 degrees Celsius, cultivates and collect supernatant liquor after 3 days; Collect the supernatant liquor of nutrient solution of 8 times of going down to posterity; Frozen for subsequent use.
Three. mixing
2. get above frozen supernatant liquor, after melting respectively, in human adipose-derived stem cell: human fibroblasts: rat hepatocytes ratio is 2.5:2.5:5 (volume ratio) mixing.
3. the mixed solution more than in 2 adds MEM basic medium in 20% ratio (volume ratio), cultivate rat bone marrow mesenchymal stem cells, 37 DEG C, CO2gas incubator (gas concentration lwevel 5%), cultivate cell attachment after 5 days, within 8-9 days, can one deck be paved with.
Claims (10)
1. prepare a method for cell cultures active ingredient compositions, said method comprising the steps of:
1) cultivate the first cell, described cell is mammal fat stem cell, is cultured to fusion rate more than 80%, and removing cell, obtains the first cell conditioned medium liquid;
2) cultivate the second cell, described cell is mammalian fibroblasts, is cultured to fusion rate more than 80%, removing cell, obtains the second cell conditioned medium liquid; And
3) by above two kinds of supernatant liquors mixing, thus described cell cultures active ingredient compositions is made.
2. the method for claim 1, is characterized in that, the volume ratio of the first cell conditioned medium liquid described and described the second cell conditioned medium liquid is 1:0.2 ~ 5.
3. the method for claim 1, it is characterized in that, described method also comprises the step of cultivating the third cell, the third cell described and stand-by described cell cultures active ingredient compositions cultured cells come from same species, be cultured to fusion rate more than 85%, removing cell, obtains the third cell conditioned medium liquid, and the three kinds of supernatant liquor mixing that will obtain, thus make described cell cultures active ingredient compositions.
4. method as claimed in claim 3, it is characterized in that, the volume ratio of the first cell conditioned medium liquid described and described the second cell conditioned medium liquid is 1:0.2 ~ 5, and the volume ratio of the first cell conditioned medium liquid described and the third cell conditioned medium liquid described is 1:0.2 ~ 5.
5. method as claimed in claim 4, it is characterized in that, the first cell described, described the second cell and the third cell described come from same Mammals respectively.
6. the method according to any one of Claims 1 to 5, it is characterized in that, also comprise and add the step that at least one is selected from following activeconstituents in supernatant liquor mixed solution: Regular Insulin, Pp63 glycophosphoproteins, linolic acid, Transferrins,iron complexes, dexamethasone, fibroblast growth factor or Sodium Selenite.
7. method as claimed in claim 6, it is characterized in that, add Regular Insulin, Pp63 glycophosphoproteins, linolic acid, Transferrins,iron complexes, dexamethasone, fibroblast growth factor, Sodium Selenite respectively in supernatant liquor mixed solution, make the final concentration of above composition be respectively 0.2-17.2ng/L, 0.2 ~ 0.8mg/mL, 0.5 ~ 2 μ g/mL, 30 ~ 70 μ g/mL, 0.025-2.54ng/L, 80 ~ 150ng/mL, 0.12-0.29ng/L.
8. a cell culture medium, comprises cell base nutritional medium and cell cultures active ingredient compositions that according to any one of claim 1 ~ 7 prepared by method.
9. cell culture medium as claimed in claim 8, it is characterized in that, the volume ratio of described cell cultures active ingredient compositions and described cell base nutritional medium is 1:5 ~ 20.
10. cell culture medium as claimed in claim 9, it is characterized in that, described cell base nutritional medium is DMEM or MEM substratum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510794654.0A CN105368769A (en) | 2015-11-18 | 2015-11-18 | Method for preparation of cell culture active component composition and cell culture medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510794654.0A CN105368769A (en) | 2015-11-18 | 2015-11-18 | Method for preparation of cell culture active component composition and cell culture medium |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105368769A true CN105368769A (en) | 2016-03-02 |
Family
ID=55371404
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510794654.0A Pending CN105368769A (en) | 2015-11-18 | 2015-11-18 | Method for preparation of cell culture active component composition and cell culture medium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105368769A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434549A (en) * | 2016-12-20 | 2017-02-22 | 江西宜信堂医疗科技有限公司 | Serum-free stem cell culture medium and preparation method thereof |
-
2015
- 2015-11-18 CN CN201510794654.0A patent/CN105368769A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| 崔红娟编著: "《干细胞生物学》", 31 May 2014, 西南师范大学出版社 * |
| 张学明等: "小鼠胚胎成纤维细胞的分离和饲养层细胞的制备", 《黑龙江畜牧兽医》 * |
| 王中兴等: "人脂肪干细胞体外培养及向软骨细胞诱导分化的实验研究", 《东南大学学报(医学版)》 * |
| 王军琳等: "大鼠骨髓间充质干细胞的体外培养与鉴定", 《实用口腔医学杂志》 * |
| 赵微等: "成人脂肪源间充质干细胞培养上清对人脐血间充质干细胞体外培养及扩增的支持作用", 《牡丹江医学院学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434549A (en) * | 2016-12-20 | 2017-02-22 | 江西宜信堂医疗科技有限公司 | Serum-free stem cell culture medium and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103555665B (en) | A kind of serum-free medium for cultivating mescenchymal stem cell | |
| CN103805562B (en) | Cultivate the serum free medium of placenta mesenchyma stem cell | |
| CN104805054B (en) | Stem cell serum-free culture medium | |
| KR101639988B1 (en) | Method for preparation of mesenchymal stem cell culture comprising high concentration of cell growth factors and composition prepared therefrom | |
| CN108251359A (en) | A kind of mesenchymal stem cell serum-free culture medium and cultural method | |
| CN106479971A (en) | A kind of serum-free medium for cultivating mescenchymal stem cell and method | |
| CN109762782B (en) | Culture medium for promoting growth of mesenchymal stem cells and preparation method thereof | |
| CN105112364A (en) | Serum-free medium for human adipose-derived stem cells and preparation method thereof | |
| Quattrocelli et al. | Mouse and human mesoangioblasts: isolation and characterization from adult skeletal muscles | |
| KR102445537B1 (en) | System and method for 3d cell culture and use thereof | |
| CN107338218A (en) | Derivant and method of a kind of induced lipolysis stem cell to Chondrocyte Differentiation | |
| RU2012110181A (en) | METHOD FOR OBTAINING AN ARTIFICIAL BODY | |
| CN102978156A (en) | Expansion in vitro purification culture method of mesenchymal stem cells and culture medium | |
| CN104988110A (en) | Method for transforming umbilical cord mesenchymal stem cells into islet cells | |
| CN104762324B (en) | Method using Runx2 and micromolecular compound induction human fibroblasts reprogramming for Gegenbaur's cell | |
| CN109943526B (en) | A kind of serum-free peptide composition promoting mescenchymal stem cell proliferation | |
| CN105112362A (en) | Serum-free medium for placenta-derived mesenchymal stem cells and preparation method thereof | |
| CN112662624A (en) | Serum-free culture medium for in vitro culture and amplification of bone marrow mesenchymal stem cells | |
| CN106754675A (en) | A kind of fat stem cell serum free medium and its production and use | |
| CN109988746A (en) | A kind of mescenchymal stem cell adipogenic induction differentiation method | |
| CN107287156A (en) | A kind of isolated culture method of fat mesenchymal stem cell and its application | |
| CN106434542A (en) | Method for enhancing proliferation and post-transplantation survival ability of adipose derived stem cells | |
| CN106367385A (en) | Culture medium and application thereof as well as method for culturing dental pulp stem cells | |
| CN103966167A (en) | Cell culture composition used for primary culture of tumor cells and application thereof | |
| CN104988111A (en) | Inducing liquid for converting UC-MSC into islet cells and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination |