CN109943525A

CN109943525A - Serum-free, non-animal derived ingredient, the specific culture medium of component and its application

Info

Publication number: CN109943525A
Application number: CN201910201406.9A
Authority: CN
Inventors: 张智勇; 任浩; 叶苗漫
Original assignee: Third Affiliated Hospital of Guangzhou Medical University
Current assignee: Zhongke Ruiji Haining Biotechnology Co ltd
Priority date: 2019-03-15
Filing date: 2019-03-15
Publication date: 2019-06-28
Anticipated expiration: 2039-03-15
Also published as: CN109943525B

Abstract

The present invention provides a kind of serum-free, non-animal derived ingredient, the specific culture medium of component and its applications, are related to technical field of cell culture.Serum-free provided by the invention, non-animal derived ingredient, the specific culture medium of component, cell factor, hormone, transferrins, albumin, myosin, putrescine and thrombocytin including basal medium and addition in the basal medium.The present invention replaces serum by adding the substances such as cell factor and hormone into basal medium, to realize the culture to people's cell.The culture medium each component substance is clear, serum-free and animal source component addition, it is lower with the lower technical problem of cell amplification efficiency that repeatability of traditional serum-containing media for cell culture be poor and other serum free mediums are cultivated cell viability can be alleviated.

Description

Serum-free, non-animal derived ingredient, the specific culture medium of component and its application

Technical field

The present invention relates to technical field of cell culture, clear more particularly, to a kind of serum-free, non-animal derived ingredient, component Culture medium and its application.

Background technique

Cell culture technology is important in RESEARCH ON CELL-BIOLOGY method and common technology, passes through analogue body inner ring in vitro Border (sterile, preference temperature, pH value and certain nutritional condition etc.), make cells survival, growth, breeding and maintain primary structure and Function obtains a large amount of cell, to study the signal transduction of cell, anabolism, growing multiplication etc..All cells from In in vitro culture, most it is difficult to people's cell culture.

Cell culture invitro needed nutrient matter in vivo it is essentially identical, for example, it is desired to sugar, amino acid, inorganic salts, growth-promoting Above-mentioned substance needed for cell is configured to culture medium strictly by its type and requirement to train by the long factor, microelement etc. Support cell.However, also need to add in traditional people's cell culture medium at present some natural animal derived components such as serum, blood plasma with One similar intracorporal environment of biology is provided.Although addition serum and the conventional medium of other animal source components can be realized people The in vitro culture of cell, but there is the substance of damage cell growth and breeding in this, such as complement, immunoglobulin and some growths Inhibiting factor, ingredient are indefinite；Repeatability of the conventional medium for cell culture is also poor, influences clinical (human body) application Safety and stability；And the serum active difference of different animals, different batches is larger, influences the stabilization of culture effect Property；Meanwhile cell viability that the conventional medium containing serum is cultivated is lower, cell amplification efficiency is lower.In order to avoid tradition The culture medium bring above problem, certain scholars by into culture medium adding ingredient determine substance replace serum and develop one Kind serum free medium, although these serum free mediums solve repeatability of the conventional medium for people's cell culture poor The problem of, the safety and stability of clinical application is improved, but the cell that its culture obtains remains cell viability Problem lower, cell amplification efficiency is lower.

In view of this, the present invention is specifically proposed.

Summary of the invention

The first purpose of this invention is to provide a kind of serum-free, non-animal derived ingredient, the specific culture medium of component, should Culture medium each component substance is clear, and non-animal derived ingredient addition, the reproducible of cell culture, cell viability are high, cell expands It is high-efficient, it is able to solve at least one of above problem.

Second object of the present invention is to provide a kind of serum-free, non-animal derived ingredient, the specific culture medium of component and exists Application in the cell culture of mesoderma origin or the cell culture of ectodermal origin, wherein the cell includes that mesenchyma is dry Cell, fibroblast, cartilage cell, neural stem cell, hematopoietic cell, myocyte, sarcoblast, endothelial cell, liver cell, One of osteocyte, osteoblast, osteoclast, nerve cell and epithelial cell are a variety of；Cell culture and experimental result It is reproducible, cell viability is high, cell amplification efficiency is high.

In order to solve the above-mentioned technical problem, the following technical scheme is adopted:

In a first aspect, the present invention provides a kind of serum-free, non-animal derived ingredient, the specific culture medium of component, including bases Cell factor in the basal medium of basal culture medium and addition, hormone, transferrins, albumin, myosin, putrescine And thrombocytin.

As further technical solution, the basal medium includes DMEM, F12, IMDM, MCDB 201 and MCDB 131 At least one of.

As further technical solution, the cell factor includes interleukins and/or growth factor；

Preferably, the concentration that the interleukins is added into the basal medium is 1~10 μ g/L；

Preferably, the growth factor include epidermal growth factor, basic fibroblast growth factor, conversion growth because At least one of son-β 1, platelet derived growth factor and insulin-like growth factor；

Preferably, the epidermal growth factor, basic fibroblast growth factor, platelet derived growth factor and pancreas The concentration that island element class growth factor is added into the basal medium is each independently 5~100ng/mL；

Preferably, the concentration that the transforming growth factor-beta 1 is added into the basal medium is 1~50ng/mL.

As further technical solution, the hormone include in insulin, hydrocortisone, epiphysin and progesterone extremely Few one kind；

Preferably, the concentration that the insulin is added into the basal medium is 5~100mg/L；

Preferably, the concentration that the hydrocortisone is added into the basal medium is 0.5~100 μ g/L；

Preferably, the concentration that the progesterone is added into the basal medium is 0.1~10 μ g/L；

Preferably, the concentration that the epiphysin is added into the basal medium is 1~100nM.

As further technical solution, concentration that the transferrins is added into the basal medium is 5~ 100mg/L；

As further technical solution, the concentration that the albumin is added into the basal medium is 1~20g/L；

As further technical solution, concentration that the myosin is added into the basal medium is 0.5~ 15g/L；

As further technical solution, the concentration that the putrescine is added into the basal medium is 0.1~20mg/L；

As further technical solution, the concentration that the thrombocytin is added into the basal medium is 1~50mg/L；

It and/or further include the fibronectin added in the basal medium；

Preferably, the concentration that the fibronectin is added into the basal medium is 10~50 μ g/L.

It further include the amino acid added in the basal medium as further technical solution；

Preferably, the amino acid includes essential amino acid and/or nonessential amino acid；

Preferably, the essential amino acid include arginine, cystine, histidine, isoleucine, leucine, lysine, At least one of methionine, phenylalanine, threonine, tryptophan, tyrosine and valine；

Preferably, the concentration that each described essential amino acid is added into the basal medium is each independently 0.01~1mM；

Preferably, the nonessential amino acid include glutamine, glycine, alanine, asparagine, aspartic acid, At least one of glutamic acid, proline and serine；

Preferably, the concentration that the glutamine is added into the basal medium is 0.5~20mM；

Preferably, the glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine are to institute It states the concentration added in basal medium and is each independently 0.01~0.2mM.

It further include the lipid added in the basal medium as further technical solution；

Preferably, the lipid includes at least one of cholesterol, tocopherol acetate and fatty acid；

Preferably, the concentration that the cholesterol is added into the basal medium is 200~1000 μ g/L；

Preferably, the concentration that the tocopherol acetate is added into the basal medium is 50~500 μ g/L；

Preferably, the fatty acid includes arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitinic acid, palm At least one of oleic acid and stearic acid；

Preferably, the concentration that the arachidonic acid is added into the basal medium is 1~50 μ g/L；

Preferably, the linoleic acid, linolenic acid, myristic acid, oleic acid, palmitinic acid, palmitoleic acid and stearic acid are to the base The concentration added in basal culture medium is each independently 10~200 μ g/L.

It further include the microelement added in the basal medium as further technical solution；

Preferably, the microelement include copper, zinc, selenium, iron, manganese, molybdenum, vanadium, nickel, tin, aluminium, silver, barium, bromine, cadmium, cobalt, At least one of chromium, fluorine, germanium, iodine, silicon, rubidium and zirconium；

Preferably, the copper, selenium, manganese, molybdenum, vanadium, nickel, tin, aluminium, silver, barium, bromine, cadmium, cobalt, chromium, fluorine, germanium, iodine, rubidium and zirconium The concentration added into the basal medium is each independently 0.01~20 μ g/L；

Preferably, the concentration that the zinc, iron and silicon are added into the basal medium is each independently 100~1200 μg/L。

It further include the vitamin added in the basal medium as further technical solution；

Preferably, the vitamin includes choline chloride, calcium pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, salt At least one of allithiamine element and inositol；

Preferably, the concentration that each described vitamin is added into the basal medium is each independently 0.1~ 2mg/L。

It further include the surfactant added in the basal medium as further technical solution；

Preferably, the surfactant includes polyoxyethylene polyoxypropylene copolymer and/or Tween 80；

Preferably, the concentration that the polyoxyethylene polyoxypropylene copolymer is added into the basal medium be 80~ 1000mg/L；

Preferably, the concentration that the Tween 80 is added into the basal medium is 2~50mg/L.

It further include the anti-oxidant protective agent added in the basal medium as further technical solution；

Preferably, the anti-oxidant protective agent includes mercaptoethanol and/or ascorbic acid；

Preferably, the concentration that the mercaptoethanol is added into the basal medium is 1~55 μM；

Preferably, the concentration that the ascorbic acid is added into the basal medium is 0.5~100mg/L.

It further include the pH buffer added in the basal medium as further technical solution；

Preferably, the pH buffer includes sodium bicarbonate and/or 4- hydroxyethyl piperazineethanesulfonic acid；

Preferably, the concentration that the sodium bicarbonate is added into the basal medium is 0.5~20g/L；

Preferably, the concentration that the 4- hydroxyethyl piperazineethanesulfonic acid is added into the basal medium is 1~10mM；

It and/or further include the catechin added in the basal medium；

Preferably, the concentration that the catechin is added into the basal medium is 10~50mg/L.

Second aspect, the present invention provides a kind of serum-free, non-animal derived ingredient, the specific culture mediums of component in mesoderm Application in the cell culture in source or the cell culture of ectodermal origin, wherein the cell include mescenchymal stem cell, at Fibrocyte, cartilage cell, neural stem cell, hematopoietic cell, myocyte, sarcoblast, endothelial cell, liver cell, osteocyte, One of osteoblast, osteoclast, nerve cell and epithelial cell are a variety of.

Compared with prior art, the present invention include it is following the utility model has the advantages that

Serum-free provided by the invention, non-animal derived ingredient, the specific culture medium of component, by adding into basal medium The substances such as refinement intracellular cytokine, hormone, transferrins, albumin, myosin, putrescine and thrombocytin replace blood serum medium, from And realize the culture to people's cell.The culture medium each component substance is clear, makes full use of the synergistic effect between each component, replaces The addition of the animal source components such as serum has preferably culture compared to traditional serum-containing media and existing serum free medium Effect can alleviate repeatable poor and other serum free medium institutes of traditional serum-containing media for cell culture The cell viability of culture is lower and the lower technical problem of cell amplification efficiency.It can be by serum-free of the invention, non-animal derived The specific culture medium of ingredient, component is applied to mescenchymal stem cell, and (including umbilical cord mesenchymal stem cells, cord blood-derived mesenchymal are dry thin Born of the same parents, mesenchymal stem cell, fat mesenchymal stem cell, endometrium mescenchymal stem cell, dental pulp mescenchymal stem cell, sheep Film mescenchymal stem cell, Synovial Mesenchymal Stem Cells, placenta mesenchyma stem cell etc.), fibroblast, cartilage cell, nerve Application in the cell culture of stem cell, hematopoietic cell and other mesoderms and ectodermal origin.

Detailed description of the invention

It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor It puts, is also possible to obtain other drawings based on these drawings.

Fig. 1 is the growth curve for the culture medium culture mescenchymal stem cell that the embodiment of the present invention 1 and comparative example 1-4 are provided (CCK-8 method).Two-way analysis of variance, *: vs.FBS p < 0.0001, vs.Opt p < 0.005, vs.Ident p < 0.0005,vs.7989 p<0.0001；**:vs.FBS p<0.0001,vs.Opt p<0.0001,vs.Ident p<0.0001, vs.7989 p<0.0001；

Fig. 2 is cell of the culture medium culture mescenchymal stem cell that provides of the embodiment of the present invention 1 and comparative example 1-4 after three days Aspect graph, scale are 50 μm (left sides) and 550 μm (right side)；

Fig. 3 is culture medium culture 3 days cells of mescenchymal stem cell that the embodiment of the present invention 1 and comparative example 1,3,5 provide Vigor (OD value (optical density value, OD value) in CCK-8 method).One-way analysis of variance, * *: vs.FBS p<0.0001,vs.Ident p<0.0001,vs.7109 p<0.0001；

Fig. 4 is that the mesenchyma in the culture medium culture Different Individual source that the embodiment of the present invention 1 and comparative example 1,3 provide is dry thin 5 days cell viabilities of born of the same parents (the OD value in CCK-8 method).One-way analysis of variance, *: vs.RZXF p of *: vs.RZXF p < 0.05, * <0.01,***:vs.RZXF p<0.001；

Fig. 5 is that the mesenchyma in the culture medium culture Different Individual source that the embodiment of the present invention 1 and comparative example 1,3 provide is dry thin Born of the same parents accumulate population doublings curve；

Fig. 6 is the mescenchymal stem cell in culture medium culture Different Individual source that provides of the embodiment of the present invention 1 to the 5th generation Karyotype.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiments and examples, but this field skill Art personnel will be understood that following embodiments and embodiment are merely to illustrate the present invention, and are not construed as limiting model of the invention It encloses.Based on the embodiments of the present invention, obtained by those of ordinary skill in the art without making creative efforts Every other embodiment, shall fall within the protection scope of the present invention.The person that is not specified actual conditions, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

It is explicitly cultivated in a first aspect, providing a kind of serum-free, non-animal derived ingredient, component at least one embodiment Base, cell factor in the basal medium, hormone, transferrins, albumin, tire ball including basal medium and addition Albumen, putrescine and thrombocytin.

Culture medium refers to supply microorganism, plant or animal (or tissue) growth and breeding, is combined by different nutriments The nutrient matrix being formulated.Generally all contain carbohydrate, nitrogen substance, inorganic salts (including microelement), vitamin With several big substances such as water.Culture medium had both been to provide cytotrophy and had promoted basic substance and the cell growth of cell Proliferation With the living environment of breeding.

Though the nutritional requirement of various cells is not identical, the basic nutrients that most cells need are identical.Basis culture Base refers to the culture medium that basic nutrients required for cell growth and breeding are prepared by.Contain cell in basal medium Basic nutrients necessary to growth and breeding can be used as the basis of some special culture mediums, further according to certain cell Particular/special requirement adds needed nutrient matter in basal medium.In the present invention, by adding other into basal medium Serum-free provided by the invention, non-animal derived ingredient, the specific culture medium of component is prepared in ingredient.

Cell factor is by immunocyte (such as monokaryon, macrophage, T cell, B cell, NK cell) and certain nonimmune One kind that cell (endothelial cell, epidermal cell, fibroblast etc.) synthesizes through stimulation, secretes has extensive biological activity Small protein.By combining the growth of corresponding receptors modulate cell, differentiation and effect, regulate and control immune response.Cell factor Interleukins, interferon, tumor necrosis factor superfamily, colony stimulating factor, chemotactic factor (CF), growth factor can be divided into Deng.In the present invention, animal blood serum is replaced by adding cell factor into basal medium, obtaining can be for human cell growth The serum-free of breeding, non-animal derived ingredient, the specific culture medium of component.

Hormone is the semiochemicals that well differentiated endocrine cell synthesized and be directly secreted into blood, it passes through adjusting Various histiocytic metabolic activities influence the physiological activity of human body.By the efficient life of endocrine gland or endocrine cells secrete Active substances transmit information as courier in vivo, play regulatory role to organism physiology process, be the important object in life Matter.In the present invention, by adding hormone into culture medium come stimulating cellular growth, cell function is maintained.

Transferrins also known as transferrin are main siderophillins in blood plasma, are mentioned for cell internalizing and cell metabolism It is that mammalian cell grows particularly important albumen for required iron.In the present invention, by being added into basal medium Transferrins is prepared serum-free, non-animal derived ingredient, the specific culture medium of component.

Albumin is most important protein in blood plasma, maintains body nutrition and osmotic pressure.Albumin has viscosity, makees The viscosity that culture medium can be increased for the ingredient of culture medium, protects cells from mechanical damage.In the present invention, by being trained to basis It supports and adds albumin in base serum-free, non-animal derived ingredient, the specific culture medium of component is prepared.

Myosin is one of to generate by liver and be secreted into blood blood protein, is had to the growth of zooblast Facilitation.In the present invention, by into basal medium add myosin come be prepared serum-free, it is non-animal derived at Divide, the specific culture medium of component.

Putrescine is one kind of biogenic amine, is widely present in cell, has the Proliferation, Differentiation of the pH and cell that adjust cell Process.In the present invention, serum-free, non-animal derived ingredient, component is prepared by adding putrescine into basal medium Specific culture medium.

Thrombocytin is a kind of transmitter substance generated in vivo, can participate in the physiological functions such as the pain sensation, sleep and body temperature Adjusting.In the present invention, by into basal medium add thrombocytin come be prepared serum-free, non-animal derived ingredient, The specific culture medium of component.

There are also some ingredients not yet to study clear, current most people's cell culture for the interior environment lived due to people's cell Need to add some natural animal derived components such as serum, blood plasma in base also to provide the similar intracorporal environment of biology.Addition Although the conventional medium of animal source component can be realized the in vitro culture of people's cell, but this has damage cell growth and numerous The substance grown, as complement, immunoglobulin and some growth inhibitory factors, ingredient are indefinite；Conventional medium is trained for cell Feeding repeatability is also poor, influences the safety and stability of clinical (human body) application；And different animals, different batches Serum active difference is larger, influences the stability of culture effect；Meanwhile conventional medium and existing free serum culture containing serum The cell viability that base is cultivated is lower, cell amplification efficiency is lower.

In the present invention, by adding cell factor, hormone, transferrins, albumin, tire ball egg into basal medium The substances such as white, putrescine and thrombocytin obtain serum-free, non-animal derived ingredient, the specific culture medium of component.The culture medium each component Substance is clear, makes full use of the synergistic effect between each component, replaces the addition of the animal source components such as serum, compared to containing serum Conventional medium and existing serum free medium, culture medium cell viability of the invention is high, cell amplification efficiency is high, has more Good culture effect can alleviate the cell culture of traditional serum-containing media and the poor repeatability of experimental result and other The low technical problem low with cell amplification efficiency of the cell viability that serum free medium is cultivated.

In a preferred embodiment, basal medium includes DMEM, F12, IMDM, MCDB 201 and MCDB 131 At least one of.

Wherein, DMEM culture medium (dulbecco's modified eagle medium) be it is a kind of containing various amino acid and The culture medium of glucose；F12 culture medium (12 nutrient medium Zooblast culture medium of Ham ' s F) complicated component, contains There are many microelements, can be used as the basis of serum free medium；IMDM culture medium (Iscove ' s Modified Dubecco ' S Medium) it is a kind of Zooblast culture medium in improvement, it is dynamic to be suitable for lymphocyte, bone marrow cell, hybridoma etc. The culture of object cell；201 culture medium of MCDB (Molecular, Cellular, and Developmental Biology) is one Kind serum free medium, can be used for the clonal growth of chicken embryo fibroblasts；131 culture medium of MCDB is a kind of free serum culture Base can be used for the culture of microvascular endothelial cells.In the present invention, it is cultivated using above-mentioned culture medium as basis of the invention Base.

Contain basic nutrients necessary to cell growth and breeding in basal medium.In the present invention, basis culture Base includes but is not limited at least one of DMEM, F12, IMDM, MCDB 201 and MCDB 131.Such as basal medium is DMEM；It or is F12；It or is IMDM；It or is MCDB 201；It or is MCDB 131；It or is DMEM and F12；Or For IMDM and MCDB 201；It or is MCDB 201 and MCDB 131；It or is DMEM, F12 and IMDM；Or for IMDM, MCDB 201 and MCDB 131；It or is DMEM, F12, IMDM and MCDB 201；It or is 201 and of F12, IMDM, MCDB MCDB 131；It or is DMEM, F12, IMDM, MCDB 201 and MCDB 131 etc.；Preferably DMEM, F12, IMDM, MCDB 201 and MCDB 131.It should be noted that it is preferred that using the above two above culture medium as basic culture medium, basis training Supporting base is above-mentioned culture medium with the mixing of arbitrary proportion.

In a preferred embodiment, cell factor includes interleukins and/or growth factor.

Interleukins is the type cytokines for generating and acting on various kinds of cell by various kinds of cell, wide variety (including interleukin 1, interleukin 2, interleukin 3 etc.), function is complicated, maturation, work in immunocyte It plays a significant role during change, proliferation and immunological regulation etc. are a series of.

Growth factor is present in organism, has the activated protein of extensive adjustment effect to growth, the development of biology Matter or polypeptides matter.Its general characteristic is can be in conjunction with cell membrane specific receptors, the work with regulating cell growth, development With to the immune of human body, the generation of hematopoietic regulation, tumour, wound healing, vascularization, cell differentiation, Apoptosis, form hair Raw, embry ogenesis etc. generates important regulating and controlling effect.Cell factor is widely present in various tissues in body, including at Ripe tissue and embryonic tissue, and adjust by autocrine and paracrine mode the proliferation and differentiation of various cells.In the present invention, Serum-free, non-animal derived ingredient, component is prepared by adding interleukins and growth factor into basal medium Specific culture medium.

Preferably, the concentration that the interleukins is added into the basal medium is 1~10 μ g/L；Leucocyte is situated between The typical but non-limiting concentration that element is added into the basal medium is 1 μ g/L, 2 μ g/L, 3 μ g/L, 4 μ g/L, 5 μ g/ L, 6 μ g/L, 7 μ g/L, 8 μ g/L, 9 μ g/L or 10 μ g/L, preferably 6 μ g/L.

In the present invention, by adding the further adjustment and optimization of concentration into basal medium to interleukins, So that the effect of the specific culture medium culture cell of serum-free provided by the invention, non-animal derived ingredient, component is more preferable.

Preferably, the growth factor includes but is not limited to epidermal growth factor, basic fibroblast growth factor, turns Change at least one of growth factor-beta 11, platelet derived growth factor and insulin-like growth factor, such as growth factor is Epidermal growth factor；It or is basic fibroblast growth factor；It or is transforming growth factor-beta 1；It or is blood platelet Derivative growth factor；It or is insulin-like growth factor；Or for epidermal growth factor, basic fibroblast growth because Son；It or is basic fibroblast growth factor, transforming growth factor-beta 1；It or is transforming growth factor-beta 1, blood platelet Derivative growth factor；It or is platelet derived growth factor and insulin-like growth factor；It or is epidermal growth factor, alkali Property fibroblast growth factor, transforming growth factor-beta 1；Or for basic fibroblast growth factor, conversion growth because Son-β 1, platelet derived growth factor；Or it is raw for transforming growth factor-beta 1, platelet derived growth factor and insulin type The long factor；It or is epidermal growth factor, basic fibroblast growth factor, transforming growth factor-beta 1, platelet-derived life The long factor；It or is basic fibroblast growth factor, transforming growth factor-beta 1, platelet derived growth factor and pancreas islet Plain class growth factor etc.；Preferably basic fibroblast growth factor, transforming growth factor-beta 1, platelet derived growth because Son and insulin-like growth factor.

In the present invention, by the adjustment and optimization to growth factor type in culture medium, so that nothing provided by the invention Serum, non-animal derived ingredient, the specific culture medium of component are more preferable to the culture effect of cell.

Preferably, the concentration that the epidermal growth factor is added into the basal medium is 5~100ng/mL.Epidermis Growth factor, basic fibroblast growth factor, platelet derived growth factor and insulin-like growth factor are to the base The typical but non-limiting concentration added in basal culture medium is each independently 5ng/mL, 10ng/mL, 20ng/mL, 30ng/ ML, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100ng/mL, preferably 50ng/mL.

Preferably, the concentration that the transforming growth factor-beta 1 is added into the basal medium is 1~50ng/mL.Turn Change the typical but non-limiting concentration added into the basal medium of growth factor-beta 11 be 1ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, 20ng/mL, 25ng/mL, 30ng/mL, 35ng/mL, 40ng/mL, 45ng/mL or 50ng/mL, preferably For 25ng/mL.

In the present invention, by adding the further adjustment and optimization of concentration into culture medium to each growth factor, make Serum-free provided by the invention, non-animal derived ingredient, the effect of the specific culture medium culture cell of component are more preferable.

In a preferred embodiment, hormone include in insulin, hydrocortisone, epiphysin and progesterone extremely Few one kind, such as hormone are insulin；It or is hydrocortisone；It or is epiphysin；It or is progesterone；It or is pancreas islet Element and hydrocortisone；It or is hydrocortisone and progesterone；It or is insulin and progesterone；It or is insulin, hydrogen Change cortisone, epiphysin and progesterone etc.；Preferably insulin, hydrocortisone, epiphysin and progesterone.

In the present invention, by the adjustment and optimization to hormone kind in culture medium, so that serum-free provided by the invention, The specific culture medium of non-animal derived ingredient, component is more preferable to the culture effect of cell.

Preferably, the concentration that the insulin is added into the basal medium is 5~100mg/L.Insulin is to institute State the typical but non-limiting concentration added in basal medium be 5mg/L, 10mg/L, 20mg/L, 30mg/L, 40mg/L, 50mg/L, 60mg/L, 70mg/L, 80mg/L, 90mg/L or 100mg/L, preferably 50mg/L.

Preferably, the concentration that the hydrocortisone is added into the basal medium is 0.5~100 μ g/L.Hydrogenation The typical but non-limiting concentration that cortisone is added into the basal medium is 0.5 μ g/L, 1 μ g/L, 10 μ g/L, 20 μ G/L, 30 μ g/L, 40 μ g/L, 50 μ g/L, 60 μ g/L, 70 μ g/L, 80 μ g/L, 90 μ g/L or 100 μ g/L, preferably 50 μ g/L.

Preferably, the concentration that the progesterone is added into the basal medium is 0.1~10 μ g/L.Progesterone is to institute Stating the typical but non-limiting concentration added in basal medium is 0.1 μ g/L, 1 μ g/L, 2 μ g/L, 3 μ g/L, 4 μ g/L, 5 μ G/L, 6 μ g/L, 7 μ g/L, 8 μ g/L, 9 μ g/L or 10 μ g/L, preferably 5 μ g/L.

Preferably, the concentration that the epiphysin is added into the basal medium is 1~100nM.Epiphysin is to described The typical but non-limiting concentration added in basal medium be 1nM, 10nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM or 100nM, preferably 50nM.

In the present invention, by adding the further adjustment and optimization of concentration into basal medium to each hormone, make Serum-free provided by the invention, non-animal derived ingredient, the effect of the specific culture medium culture cell of component are more preferable.

In a preferred embodiment, the concentration that transferrins is added into the basal medium be 5~ 100mg/L.The typical but non-limiting concentration that transferrins is added into the basal medium be 5mg/L, 10mg/L, 20mg/L, 30mg/L, 40mg/L, 50mg/L, 60mg/L, 70mg/L, 80mg/L, 90mg/L or 100mg/L, preferably 50mg/ L。

In a preferred embodiment, the concentration that albumin is added into the basal medium is 1~20g/L. The typical but non-limiting concentration that albumin is added into the basal medium be 1g/L, 2g/L, 4g/L, 6g/L, 8g/L, 10g/L, 12g/L, 14g/L, 16g/L, 18g/L or 20g/L, preferably 10g/L.

In a preferred embodiment, the concentration that myosin is added into the basal medium be 0.5~ 15g/L.The typical but non-limiting concentration that myosin is added into the basal medium be 0.5g/L, 1g/L, 2g/L, 4g/L, 6g/L, 8g/L, 10g/L, 12g/L, 14g/L or 15g/L, preferably 8g/L.

In a preferred embodiment, the concentration that putrescine is added into the basal medium is 0.1~20mg/L. The typical but non-limiting concentration that putrescine is added into the basal medium be 0.1mg/L, 1mg/L, 2mg/L, 4mg/L, 6mg/L, 8mg/L, 10mg/L, 12mg/L, 14mg/L, 16mg/L, 18mg/L or 20mg/L, preferably 10mg/L.

In a preferred embodiment, the concentration that thrombocytin is added into the basal medium is 1~50mg/L. The typical but non-limiting concentration that thrombocytin is added into the basal medium is 1mg/L, 5mg/L, 15mg/L, 20mg/ L, 25mg/L, 30mg/L, 35mg/L, 40mg/L, 45mg/L or 50mg/L, preferably 25mg/L.

It in a preferred embodiment, further include the fibronectin added in the basal medium.

Preferably, the concentration that the fibronectin is added into the basal medium is 10~50 μ g/L.Fiber The typical but non-limiting concentration that connection albumen is added into the basal medium is 10 μ g/L, 15 μ g/L, 20 μ g/L, 25 μ g/L, 30 μ g/L, 35 μ g/L, 40 μ g/L, 45 μ g/L, 50 μ g/L, preferably 30 μ g/L.

In the present invention, by the way that said components are added with the further adjustment and optimization of concentration into culture medium, so that this It is more preferable to invent the serum-free, non-animal derived ingredient, the effect of the specific culture medium culture cell of component provided.

It in a preferred embodiment, further include the amino acid added in the basal medium.

Amino acid contains basic amine group and acidic carboxypolymer, is the base substance of protein needed for constituting Animal nutrition.It is training Carbon source and nitrogen source needed for addition amino acid can provide its growth and breeding of maintenance in feeding base for cell.

Preferably, the amino acid includes essential amino acid and/or nonessential amino acid.Such as amino acid is；Or it is Nonessential amino acid；It or is essential amino acid and nonessential amino acid；Preferably essential amino acid and nonessential amino acid.

Preferably, the essential amino acid include arginine, cystine, histidine, isoleucine, leucine, lysine, At least one of methionine, phenylalanine, threonine, tryptophan, tyrosine and valine.Such as essential amino acid is essence Propylhomoserin；It or is cystine；It or is histidine；It or is isoleucine；It or is arginine and cystine；It or is group ammonia Acid and isoleucine；It or is leucine and lysine；It or is arginine, cystine and histidine；Or for isoleucine, Leucine and lysine；It or is arginine, cystine, histidine and isoleucine；It or is leucine, lysine, first sulphur Propylhomoserin and phenylalanine；It or is arginine, cystine, histidine, isoleucine, leucine, lysine, methionine, benzene Alanine, threonine, tryptophan, tyrosine and valine etc.；Preferably arginine, cystine, histidine, isoleucine, bright Propylhomoserin, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and valine.

Preferably, the concentration that each described essential amino acid is added into the basal medium is each independently 0.01~1mM.

Wherein, the concentration of arginine addition is 0.4~0.8mM；The concentration of cystine addition is 0.01~0.2mM；Group ammonia The concentration of acid addition is 0.1~0.4mM；The concentration of isoleucine addition is 0.2~0.6mM；The concentration of leucine addition is 0.2 ~0.6mM；The concentration of lysine addition is 0.2~0.6mM；The concentration of methionine addition is 0.01~0.2mM；Phenylalanine The concentration of addition is 0.1~0.4mM；The concentration of threonine addition is 0.2~0.6mM；Tryptophan addition concentration be 0.01~ 0.1mM；The concentration of tyrosine addition is 0.1~0.4mM；The concentration of valine addition is 0.2~0.6mM.

Preferably, the nonessential amino acid include glutamine, glycine, alanine, asparagine, aspartic acid, At least one of glutamic acid, proline and serine.Such as nonessential amino acid is glutamine；It or is glycine；Or For alanine；It or is asparagine；It or is aspartic acid；It or is glutamic acid；It or is proline；It or is silk ammonia Acid；It or is glutamine and glycine；It or is alanine and asparagine；It or is aspartic acid and glutamic acid；Or it is Proline and serine；It or is glutamine, glycine and alanine；It or is glutamic acid, proline and serine；Or For aspartic acid, glutamic acid, proline and serine；It or is glutamine, glycine, alanine and asparagine；Or it is Glutamine, glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine etc.；Preferably glutamy Amine, glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine.

Preferably, the concentration that the glutamine is added into the basal medium is 0.5~20mM.Glutamine to The typical but non-limiting concentration added in the basal medium be 0.5mM, 1mM, 2mM, 4mM, 6mM, 8mM, 10mM, 12mM, 14mM, 16mM, 18mM or 20mM, preferably 10mM.

Preferably, the glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine are to institute It states the concentration added in basal medium and is each independently 0.01~0.2mM.Wherein, each nonessential amino acid is to described The typical but non-limiting concentration added in basal medium be each independently 0.01mM, 0.02mM, 0.04mM, 0.06mM, 0.08mM, 0.1mM, 0.12mM, 0.14mM, 0.16mM, 0.18mM or 0.2mM, preferably 0.1mM.

In the present invention, addition concentration by the type to amino acid and into culture medium carries out further adjustment and excellent Change, so that the effect of the specific culture medium culture cell of serum-free provided by the invention, non-animal derived ingredient, component is more preferable.

It in a preferred embodiment, further include the lipid added in the basal medium；

Lipid is one of the important nutrient that human body needs, energy needed for supplying body, provide it is required needed for body Fatty acid is the constituent of cell tissue；Fatty acid is a kind of monoester of letter, it be many more complicated rouge composition at Point.Energy can be provided for cell.In the present invention, lipid and fatty acid are added into culture medium to maintain the health of cell raw It is long.

Preferably, the lipid includes cholesterol and/or tocopherol acetate, such as lipid is cholesterol；Or it makes a living Educate phenol acetic acid esters；It or is cholesterol and tocopherol acetate etc.；Preferably cholesterol and tocopherol acetate.

Preferably, the concentration that the cholesterol is added into the basal medium is 200~1000 μ g/L.Cholesterol to The typical but non-limiting concentration added in the basal medium be 200 μ g/L, 250 μ g/L, 300 μ g/L, 350 μ g/L, 400μg/L、450μg/L、500μg/L、550μg/L、600μg/L、650μg/L、700μg/L、750μg/L、800μg/L、850μ G/L, 900 μ g/L, 950 μ g/L or 1000 μ g/L, preferably 600 μ g/L.

Preferably, the concentration that the tocopherol acetate is added into the basal medium is 50~500 μ g/L.Fertility The typical but non-limiting concentration that phenol acetic acid esters is added into the basal medium be 50 μ g/L, 100 μ g/L, 150 μ g/L, 200 μ g/L, 250 μ g/L, 300 μ g/L, 350 μ g/L, 400 μ g/L, 450 μ g/L or 500 μ g/L, preferably 250 μ g/L.

Preferably, the fatty acid includes arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitinic acid, palm At least one of oleic acid and stearic acid, such as fatty acid are arachidonic acid；It or is linoleic acid；It or is linolenic acid；Or Person is myristic acid；It or is oleic acid；It or is palmitinic acid；It or is palmitoleic acid；It or is stearic acid；It or is arachidonic Acid, linoleic acid；It or is linolenic acid, cardamom pinolenic acid, palmitinic acid；It or is arachidonic acid, linoleic acid, linolenic acid, cardamom Acid；It or is arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitinic acid, palmitoleic acid and stearic acid etc.；It is preferred that For arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitinic acid, palmitoleic acid and stearic acid.

Preferably, the concentration that the arachidonic acid is added into the basal medium is 1~50 μ g/L.Arachidonic The typical but non-limiting concentration that acid is added into the basal medium is 1 μ g/L, 5 μ g/L, 10 μ g/L, 15 μ g/L, 20 μ G/L, 25 μ g/L, 30 μ g/L, 35 μ g/L, 40 μ g/L, 45 μ g/L or 50 μ g/L, preferably 25 μ g/L.

Preferably, the linoleic acid, linolenic acid, myristic acid, oleic acid, palmitinic acid, palmitoleic acid and stearic acid are to the base The concentration added in basal culture medium is each independently 10~200 μ g/L.Linoleic acid, linolenic acid, myristic acid, oleic acid, palmitinic acid, The typical but non-limiting concentration that palmitoleic acid and stearic acid are added into the basal medium is each independently 10 μ g/ L, 20 μ g/L, 40 μ g/L, 60 μ g/L, 80 μ g/L, 100 μ g/L, 120 μ g/L, 140 μ g/L, 160 μ g/L, 180 μ g/L or 200 μ g/ L, preferably 100 μ g/L.

In the present invention, the addition concentration by the type to lipid and fatty acid and into culture medium is further adjusted Whole and optimization, so that the effect of the specific culture medium culture cell of serum-free provided by the invention, non-animal derived ingredient, component is more It is good.

It in a preferred embodiment, further include the microelement added in the basal medium.

Microelement is often referred to that content in human body is few but indispensable minerals, generally below body weight 0.01%.In cell cultivation process, microelement participates in the growth of cell and the generation of product, constitutes the knot of tissue of cell Structure.

Preferably, the microelement include copper, zinc, selenium, iron, manganese, molybdenum, vanadium, nickel, tin, aluminium, silver, barium, bromine, cadmium, cobalt, At least one of chromium, fluorine, germanium, iodine, silicon, rubidium and zirconium, such as microelement are copper；It or is zinc；It or is silicon；Or for selenium and Iron；It or is manganese and molybdenum；It or is vanadium, nickel and tin；It or is aluminium, silver, barium and bromine；It or is cadmium, cobalt, chromium, fluorine and germanium；Or For chromium, fluorine, germanium, iodine, silicon, rubidium and zirconium；Or for copper, zinc, selenium, iron, manganese, molybdenum, vanadium, nickel, tin, aluminium, silver, barium, bromine, cadmium, cobalt, Chromium, fluorine, germanium, iodine, silicon, rubidium and zirconium etc.；Preferably copper, zinc, selenium, iron, manganese, molybdenum, vanadium, nickel, tin, aluminium, silver, barium, bromine, cadmium, cobalt, Chromium, fluorine, germanium, iodine, silicon, rubidium and zirconium.

Wherein, the concentration of copper addition is 0.1~2 μ g/L；The concentration of Zn-ef ficiency addition is 100~900 μ g/L；Selenium member The concentration of element addition is 0.5~20 μ g/L；The concentration of ferro element addition is 100~1200 μ g/L；Manganese element addition concentration be 0.01~0.2 μ g/L；The concentration of molybdenum element addition is 0.1~1.5 μ g/L；The concentration of vanadium addition is 0.05~0.9 μ g/L； The concentration of nickel element addition is 0.01~0.2 μ g/L；The concentration of tin element addition is 0.01~0.2 μ g/L；Aluminium element addition Concentration is 0.1~1.5 μ g/L；The concentration of silver element addition is 0.01~0.2 μ g/L；The concentration of barium element addition is 0.5~20 μ g/L；The concentration of bromo element addition is 0.01~0.2 μ g/L；The concentration of cadmium element addition is 0.5~20 μ g/L；Cobalt element addition Concentration be 0.5~20 μ g/L；The concentration of chromium addition is 0.05~0.9 μ g/L；The concentration of fluorine element addition is 0.5~20 μg/L；The concentration of Germanium addition is 0.1~1.5 μ g/L；The concentration of iodine addition is 0.05~0.9 μ g/L；Element silicon adds The concentration added is 110~1000 μ g/L；The concentration of rubidium element addition is 0.1~1.5 μ g/L；The concentration of zr element addition is 0.5 ~20 μ g/L.

In the present invention, addition concentration by the type to microelement and into culture medium carry out further adjustment and Optimization, so that the effect of the specific culture medium culture cell of serum-free provided by the invention, non-animal derived ingredient, component is more preferable.

It in a preferred embodiment, further include the vitamin added in the basal medium.

Vitamin is that humans and animals are to maintain normal physiological function and must obtain from food a kind of micro organic Substance plays an important role in growth in humans, metabolism, growth course.In the present invention, the addition dimension life into culture medium Element is to maintain the healthy growth of cell.

Preferably, the vitamin includes choline chloride, calcium pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, salt At least one of allithiamine element and inositol, such as vitamin are choline chloride；It or is calcium pantothenate；It or is folic acid；Or For niacinamide；It or is pyridoxal hydrochloride and riboflavin；It or is thiamine hydrochloride and inositol；It or is choline chloride, pantothenic acid Calcium and folic acid；It or is niacinamide, pyridoxal hydrochloride, riboflavin and thiamine hydrochloride；It or is choline chloride, calcium pantothenate, leaf Acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride and inositol etc.；Preferably choline chloride, calcium pantothenate, folic acid, cigarette Amide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride and inositol.

Wherein, the concentration that choline chloride, calcium pantothenate, folic acid, niacinamide, pyridoxal hydrochloride and thiamine hydrochloride add is respectively It independently is 0.5~1.5mg/L；The concentration of riboflavin addition is 0.1~1mg/L；The concentration of inositol addition is 1~2mg/L.

In the present invention, addition concentration by the type to vitamin and into culture medium carries out further adjustment and excellent Change, so that the effect of the specific culture medium culture cell of serum-free provided by the invention, non-animal derived ingredient, component is more preferable.

It in a preferred embodiment, further include the surfactant added in the basal medium.

The surfactant for adding suitable concentration in the medium changes cell membrane permeability, enhancing substrate and oxygen and passes Matter promotes growth, improves the conversion ratio of substrate.

Preferably, the surfactant includes polyoxyethylene polyoxypropylene copolymer and/or Tween 80, for example, polyoxy Propylene polyoxyethylene copolymer；It or is Tween 80；It or is polyoxyethylene polyoxypropylene copolymer and Tween 80 etc.；Preferably Polyoxyethylene polyoxypropylene copolymer.

Preferably, the concentration that the polyoxyethylene polyoxypropylene copolymer is added into the basal medium be 80~ 1000mg/L.The typical but non-limiting concentration that polyoxyethylene polyoxypropylene copolymer is added into the basal medium is 800mg/L、100mg/L、200mg/L、300mg/L、400mg/L、500mg/L、600mg/L、700mg/L、800mg/L、 900mg/L or 1000mg/L, preferably 500mg/L.

Preferably, the concentration that the Tween 80 is added into the basal medium is 2~50mg/L.Tween 80 is to described The typical but non-limiting concentration added in basal medium be 2mg/L, 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L, 30mg/L, 35mg/L, 40mg/L, 45mg/L or 50mg/L, preferably 25mg/L.

In the present invention, the addition concentration by the type of Surfactant and into culture medium is further adjusted And optimization, so that the effect of the specific culture medium culture cell of serum-free provided by the invention, non-animal derived ingredient, component is more It is good.

It in a preferred embodiment, further include the anti-oxidant protective agent added in the basal medium.

In the present invention, by adding anti-oxidant protective agent into culture medium to prevent the rotten of effective component in culture medium Failure, extends the service life of culture medium.

Preferably, the anti-oxidant protective agent includes mercaptoethanol and/or ascorbic acid, such as anti-oxidant protective agent is mercapto Base ethyl alcohol；It or is ascorbic acid；It or is mercaptoethanol and ascorbic acid etc.；Preferably mercaptoethanol.

Preferably, the concentration that the mercaptoethanol is added into the basal medium is 1~55 μM.Mercaptoethanol is to institute Stating the typical but non-limiting concentration added in basal medium is 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM or 55 μM, preferably 25 μM.

Preferably, the concentration that the ascorbic acid is added into the basal medium is 0.5~100mg/L.Vitamin C The typical but non-limiting concentration that acid is added into the basal medium be 0.5mg/L, 1mg/L, 10mg/L, 20mg/L, 30mg/L, 40mg/L, 50mg/L, 60mg/L, 70mg/L, 80mg/L, 90mg/L or 100mg/L, preferably 50mg/L.

In the present invention, the addition concentration by the type to anti-oxidant protective agent and into culture medium is further adjusted Whole and optimization, so that the effect of the specific culture medium culture cell of serum-free provided by the invention, non-animal derived ingredient, component is more It is good.

It in a preferred embodiment, further include the pH buffer added in the basal medium.

In the present invention, by adding pH buffer into culture medium, maintain the pH of culture medium in a stable range It is interior, to realize the better growth of cell.

Preferably, the pH buffer includes sodium bicarbonate and/or 4- hydroxyethyl piperazineethanesulfonic acid, such as pH buffer is Sodium bicarbonate；It or is 4- hydroxyethyl piperazineethanesulfonic acid；It or is sodium bicarbonate and 4- hydroxyethyl piperazineethanesulfonic acid etc.；Preferably Sodium bicarbonate.

Preferably, the concentration that the sodium bicarbonate is added into the basal medium is 0.5~20g/L.Sodium bicarbonate The typical but non-limiting concentration added into the basal medium is 0.5g/L, 2g/L, 4g/L, 6g/L, 8g/L, 10g/ L, 12g/L, 14g/L, 16g/L, 18g/L or 20g/L, preferably 10g/L.

Preferably, the concentration that the 4- hydroxyethyl piperazineethanesulfonic acid is added into the basal medium is 1~10mM.4- The typical but non-limiting concentration that hydroxyethyl piperazineethanesulfonic acid is added into the basal medium be 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM or 10mM, preferably 5mM.

In the present invention, addition concentration by the type to pH buffer and into culture medium carry out further adjustment and Optimization, so that the effect of the specific culture medium culture cell of serum-free provided by the invention, non-animal derived ingredient, component is more preferable.

It in a preferred embodiment, further include the catechin added in the basal medium.

Preferably, the concentration that the catechin is added into the basal medium is 10~50mg/L.Catechin is to institute State the typical but non-limiting concentration added in basal medium be 10mg/L, 15mg/L, 20mg/L, 25mg/L, 30mg/L, 35mg/L, 40mg/L, 45mg/L or 50mg/L, preferably 30mg/L.

In the present invention, by the way that addition concentration of the catechin into basal medium is further adjusted and optimized, So that the effect of the specific culture medium culture cell of serum-free provided by the invention, non-animal derived ingredient, component is more preferable.

As a kind of preferred embodiment of the invention, the serum-free, non-animal derived ingredient, component are explicitly cultivated Base, including basal medium, and add cell factor in the basal medium, hormone, transferrins, albumin, Myosin, putrescine, thrombocytin, fibronectin, amino acid, lipid, microelement, vitamin, surfactant, antioxygen Change protective agent, pH buffer and catechin.

Second aspect, the present invention provides a kind of serum-free, non-animal derived ingredient, the specific culture mediums of component in mesenchyma Stem cell, fibroblast, cartilage cell, neural stem cell, hematopoietic cell and other mesoderms and ectodermal origin cell Application in culture.

In the present invention, by adding cell factor, hormone, transferrins, albumin, tire ball egg into basal medium The substances such as white, putrescine and thrombocytin obtain serum-free, non-animal derived ingredient, the specific culture medium of component.The culture medium each component Substance is clear, makes full use of the synergistic effect between each component, replaces the addition of the animal source components such as serum, by training of the invention Base is supported to be applied to mescenchymal stem cell (including umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, medulla mesenchyma are dry thin Born of the same parents, fat mesenchymal stem cell, endometrium mescenchymal stem cell, dental pulp mescenchymal stem cell, amnion mesenchymal stem cell, cunning Film mescenchymal stem cell, placenta mesenchyma stem cell etc.), fibroblast, cartilage cell, neural stem cell, hematopoietic cell and The cell culture of other mesoderms and ectodermal origin can be obtained compared to traditional serum-containing media and existing culture medium Better culture effect can alleviate repeatable poor and other no blood of traditional serum-containing media for cell culture The cell viability that clear culture medium is cultivated is lower and the lower technical problem of cell amplification efficiency.

The present invention is further illustrated below by specific embodiment and comparative example, it should be understood, however, that, these implementations Example, which is only used for being described in more detail, to be used, and but should not be understood as present invention is limited in any form.

Embodiment 1

Present embodiments provide a kind of serum-free, non-animal derived ingredient, the specific culture medium of component (RZXF), each component Content is as shown in table 1.

Serum-free, the non-animal derived ingredient, the specific culture medium of component of 1 embodiment of the present invention 1 of table offer

Note: each material concentration is the concentration additionally added in table, without the concentration for having substance in basal medium.

Comparative example 1

This comparative example provides a kind of traditional 10% fetal calf serum DMEM/F12 mammalian cell culture (FBS).

Comparative example 2

This comparative example provides a kind of serum free medium (Opt), is " Rajaraman G, White J, Tan K S, et al.Optimization and scale-up culture of human endometrial multipotent mesenchymal stromal cells:potential for clinical application[J].Tissue Engineering Part C:Methods, 2012,19 (1): serum free medium used in 80-92. ".

Comparative example 3

This comparative example provides a kind of serum free medium (Ident), is " Jung S, Sen A, Rosenberg L, et al.Identification of growth and attachment factors for the serum-free isolation and expansion of human mesenchymal stromal cells[J].Cytotherapy, 2010,12 (5): serum free medium used in 637-657. ".

Comparative example 4

This comparative example provides a kind of serum free medium (7989), is the training of serum-free used in United States Patent (USP) US7989205 Support base.

Comparative example 5

This comparative example provides a kind of serum free medium (7109), is the training of serum-free used in United States Patent (USP) US7109032 Support base.

Test example 1

Mescenchymal stem cell (MSC) is inoculated in respectively in the culture medium of embodiment 1 and comparative example 1-5 offer and is trained It supports.It is detected using vigor of the CCK-8 method to the mescenchymal stem cell in each culture medium, vigor is indicated with OD value, knot Fruit is as shown in Figs. 1-5.

Culture medium culture mescenchymal stem cell the 3rd, 6,9 day OD value such as Fig. 1 institute that embodiment 1 and comparative example 1-4 are provided Show.From figure 1 it appears that the growth of mescenchymal stem cell in RZXF, 7989 and FBS culture medium with incubation time, carefully The vigor of born of the same parents gradually rises；With the growth of incubation time, the vigor of cell first increased for cell in Opt and Ident culture medium After reduce, wherein the vigor of cell is substantially higher the vigor of the cell in other comparative example cultures in RZXF culture medium, and cell expands Increasing Efficiency is high, illustrates that culture medium culture effect provided by the invention is best.

Fig. 2 is cell of the culture medium culture mescenchymal stem cell that provides of the embodiment of the present invention 1 and comparative example 1-4 after three days Aspect graph.By culture in three days, mescenchymal stem cell can mass propagation in the medium, the culture that wherein embodiment 1 provides The mescenchymal stem cell quantity that base culture obtains is maximum, and cellular morphology is more visible obviously, three-dimensional sense is strong；7989 culture medium cultures Obtained mescenchymal stem cell quantity is minimum, can obtain in conjunction with Fig. 1, Fig. 2, the quantity of mescenchymal stem cell and cell in culture medium Vigor is positively correlated, and therefore, culture medium culture effect provided by the invention is best.

3 days OD values of culture medium culture mescenchymal stem cell that embodiment 1 and comparative example 1,3,5 provide as shown in figure 3, from As can be seen that the OD value of the RZSF culture medium culture mescenchymal stem cell of offer of the invention is apparently higher than other types in Fig. 3 Culture medium, illustrate that the cell viability that culture medium culture provided by the invention obtains is high.

Embodiment 1 and comparative example 1,3 culture medium cultures Different Individual source mescenchymal stem cell 5 days OD values as schemed Shown in 4.Wherein MSC1, MSC2 and MSC3 are respectively the mescenchymal stem cell in three Different Individual sources.From fig. 4, it can be seen that Serum-free of the invention, non-animal derived ingredient, the vigor of cell is substantially higher in Ident and FBS in the specific culture medium of component The vigor of cell in culture illustrates that culture medium provided by the invention is trained compared to traditional serum-containing media and existing serum-free The culture to people's cell can be better achieved in feeding base.And each component substance is clear in the present invention, serum-free and animal sources at Divide addition, repeatable poor and other serum free mediums of traditional serum-containing media for cell culture can be alleviated The cell viability cultivated is lower and the lower technical problem of cell amplification efficiency.

The mescenchymal stem cell in the culture medium culture Different Individual source that embodiment 1 and comparative example 1,3 provide accumulates group The curve that doubles is as shown in Figure 5.Wherein MSC1, MSC2 and MSC3 are respectively the mescenchymal stem cell in three Different Individual sources.From It can be seen that the increase with cultivated days, the accumulation cell population doublings of three culture medium culture mescenchymal stem cells in figure Value increases, and then tends towards stability, wherein the accumulation cell population doublings value in RXSF culture medium each stage be above other two A culture, the amplification efficiency highest for the mescenchymal stem cell that RXSF culture medium culture culture obtains, culture effect are best.

Test example 2

The mescenchymal stem cell in Different Individual source is inoculated in the culture medium (RZXF) that embodiment 1 provides and is trained It supports, chromosome karyotype analysis is carried out to the cell of culture to the 5th generation, as a result as shown in fig. 6, wherein MSC1, MSC2 and MSC3 points Not Wei three Different Individual sources mescenchymal stem cell.

It can be seen from the figure that being incited somebody to action using serum-free provided by the invention, non-animal derived ingredient, the specific culture medium of component After cell culture to 5 generations, cell dye-free body is abnormal.Illustrate that culture medium of the invention is able to maintain that cell normal chromosomal core Type, culture medium of the invention can be applied to the culture of the people's cells such as mescenchymal stem cell by this well.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution The range of scheme.

Claims

1. a kind of serum-free, non-animal derived ingredient, the specific culture medium of component, which is characterized in that including basal medium and add Cell factor, hormone, transferrins, albumin, myosin, putrescine and the thrombocytin being added in the basal medium.

2. serum-free according to claim 1, non-animal derived ingredient, the specific culture medium of component, which is characterized in that described Basal medium includes at least one of DMEM, F12, IMDM, MCDB 201 and MCDB 131.

3. serum-free according to claim 1, non-animal derived ingredient, the specific culture medium of component, which is characterized in that described Cell factor includes interleukins and/or growth factor；

Preferably, the growth factor includes epidermal growth factor, basic fibroblast growth factor, transforming growth factor-β 1, at least one of platelet derived growth factor and insulin-like growth factor；

Preferably, the epidermal growth factor, basic fibroblast growth factor, platelet derived growth factor and insulin The concentration that class growth factor is added into the basal medium is each independently 5~100ng/mL；

4. serum-free according to claim 1, non-animal derived ingredient, the specific culture medium of component, which is characterized in that described Hormone includes at least one of insulin, hydrocortisone, epiphysin and progesterone；

5. serum-free according to claim 1, non-animal derived ingredient, the specific culture medium of component, which is characterized in that described The concentration that transferrins is added into the basal medium is 5~100mg/L；

And/or the concentration that the albumin is added into the basal medium is 1~20g/L；

And/or the concentration that the myosin is added into the basal medium is 0.5~15g/L；

And/or the concentration that the putrescine is added into the basal medium is 0.1~20mg/L；

And/or the concentration that the thrombocytin is added into the basal medium is 1~50mg/L；

It and/or further include the fibronectin added in the basal medium；

6. serum-free according to claim 1, non-animal derived ingredient, the specific culture medium of component, which is characterized in that also wrap The amino acid added in the basal medium is included, the amino acid includes essential amino acid and/or nonessential amino acid；

Preferably, the essential amino acid includes arginine, cystine, histidine, isoleucine, leucine, lysine, first sulphur At least one of propylhomoserin, phenylalanine, threonine, tryptophan, tyrosine and valine, each described essential amino acid to The concentration added in the basal medium is each independently 0.01~1mM；

Preferably, the nonessential amino acid includes glutamine, glycine, alanine, asparagine, aspartic acid, paddy ammonia At least one of acid, proline and serine；

Preferably, the glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine are to the base The concentration added in basal culture medium is each independently 0.01~0.2mM.

7. serum-free according to claim 1, non-animal derived ingredient, the specific culture medium of component, which is characterized in that also wrap Include and add lipid in the basal medium, the lipid include in cholesterol, tocopherol acetate and fatty acid extremely Few one kind；

Preferably, the fatty acid includes arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitinic acid, palmitoleic acid At least one of with stearic acid；

Preferably, the linoleic acid, linolenic acid, myristic acid, oleic acid, palmitinic acid, palmitoleic acid and stearic acid are trained to the basis It supports the concentration added in base and is each independently 10~200 μ g/L.

8. serum-free according to claim 1, non-animal derived ingredient, the specific culture medium of component, which is characterized in that also wrap Include and add microelement in the basal medium, the microelement include copper, zinc, selenium, iron, manganese, molybdenum, vanadium, nickel, At least one of tin, aluminium, silver, barium, bromine, cadmium, cobalt, chromium, fluorine, germanium, iodine, silicon, rubidium and zirconium；

Preferably, the copper, selenium, manganese, molybdenum, vanadium, nickel, tin, aluminium, silver, barium, bromine, cadmium, cobalt, chromium, fluorine, germanium, iodine, rubidium and zirconium are to institute It states the concentration added in basal medium and is each independently 0.01~20 μ g/L；

Preferably, the concentration that the zinc, iron and silicon are added into the basal medium is each independently 100~1200 μ g/ L；

And/or further include the vitamin added in the basal medium, the vitamin include choline chloride, calcium pantothenate, At least one of folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride and inositol, each described vitamin to The concentration added in the basal medium is each independently 0.1~2mg/L.

9. serum-free according to claim 1-8, non-animal derived ingredient, the specific culture medium of component, feature It is, further includes the surfactant added in the basal medium；

Preferably, the concentration that the Tween 80 is added into the basal medium is 2~50mg/L；

It and/or further include the anti-oxidant protective agent added in the basal medium；

Preferably, the concentration that the ascorbic acid is added into the basal medium is 0.5~100mg/L；

It and/or further include the pH buffer added in the basal medium；

It and/or further include the catechin added in the basal medium；

10. the described in any item serum-frees of claim 1-9, non-animal derived ingredient, the specific culture medium of component come in mesoderm Application in the cell culture in source or the cell culture of ectodermal origin, which is characterized in that the cell includes that mesenchyma is dry thin Born of the same parents, fibroblast, cartilage cell, neural stem cell, hematopoietic cell, myocyte, sarcoblast, endothelial cell, liver cell, bone One of cell, osteoblast, osteoclast, nerve cell and epithelial cell are a variety of.