CN104099294A - Medium based on stem cell-excreted factor and its preparation method and use method - Google Patents
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Abstract
The invention discloses a medium based on a stem cell-excreted bioactive factor and its preparation method and use method. The preparation method comprises collecting a waste culture solution replaced in a stem cell culture amplification process, carrying out freeze thawing, low-temperature separation, ultrafiltration and condensation on the waste culture solution to obtain a solution rich in abundant bioactive factors, determining a protein concentration of the concentrate and adding the concentrate into a base medium according to a certain ratio. The medium obtained by the preparation method can be used for culture of cells such as various mesenchymal stem cells, human umbilical vein endothelial cells and human normal hepatocytes.
Description
Technical field
The invention belongs to biological technical field, be specifically related to collect stem cell nutrient solution supernatant, the biologically active substance of secreting in centrifugal concentrating Stem cells cultured in vitro amplification procedure is also applied.
Background technology
Stem cell is the early stage undifferentiated multipotential cell with self-replacation renewal, highly propagation and Multidirectional Differentiation of a class, and in recent years, stem cell is transitioned into clinical from laboratory study gradually.At present comprise embryonic stem cell, hemopoietic stem cell, mescenchymal stem cell (mesenchymal stem cell, MSC) and endothelial progenitor cells etc. for clinical transplanting stem cell type.MSC has multi-lineage potential, not only can be divided into hematopoietic stromal cell, can also be to mesoblastema differentiation such as scleroblast, chondrocyte, adipocyte, Tenocyte cell, myocardial cell, stroma cell, endotheliocyte, liver and biliary tract epithelial cell, pulmonary epithelial cellses, the potentiality that oriented ectodermic neuronal cell and endoblastic elliptocyte break up again simultaneously.As a kind of tissue stem cell, MSC not only has the general character of stem cell, also has its unique feature: wide material sources, be easy to vitro culture, there is the amplification ability of height and can keep good gene stability, can combine with various virus vector, the probability of immunological rejection is low etc.Therefore, increasingly mature along with MSC and correlation technique thereof, MSC be it is believed that it is desirable seed cell, a large amount of is applied to experiment and clinical study.
Previously think, stem cell is divided into target tissue cell (as cardiac muscle and endotheliocyte, i.e. transdifferentiationof) and performance therapeutic action mainly as seed cell by after transplanting.But differentiation of stem cells, particularly directed differentiation depends on surrounding environment.Increasing evidence shows that stem cell is not only the Differentiation of stem cell to histoorgan reparation and provide protection thereof, and the biologically active substance of stem cell paracrine also plays an important role.Stem cell can produce a series of somatomedins or part in residing specific microenvironment, as interleukin (IL), stem cell factor (SCF), granulocyte-macrophage colony stimutaing factor (GM-CSF), transforming growth factor-beta (TGF-β), Urogastron (EGF), fibroblast growth factor (FGF), pHGF (HGF), rhIGF-1 (IGF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF) etc., MSC participates in hematopoiesis support by these factors, the functions such as immunomodulatory, these factors also can interact with MSC simultaneously, maintain the propagation of stem cell, upgrade and differentiation.Marrow MSC can secrete some hematopoiesis correlation factors, as IL-6, IL-7, IL-8, IL-11, IL-12, IL-14, IL-15, leukaemia inhibitory factor (ILF), GM-GSF and SCF performance Hematopoiesis Support affect.Some researchs have also confirmed that MSC can also secrete TGF-β, VEGF, platelet-derived somatomedin (PDGF), keratinocyte growth factor (KGF) and HGF under certain condition stimulates.The active substance of stem cell secretion affects vascular cell and breeds, moves, sticks and multiple links of the vasculogenesis such as extracellular matrix formation.Multiple research is found, plant in the MSC of liver and secrete cytokine profiles and somatomedin by paracrine form, if IL-10, TNF, GM-CSF, HGF and NGF etc. are in promoting liver regeneration and reparation, suppress inflammation reaction, promote extracellular mechanism (ECM) to degrade, improve the liver injury of apoptosis induction, improve serum albumin level etc.IGF-1, VEGF, HGF etc. have the effect of anti-angiogenic endothelium and hepatocellular apoptosis; And HGF is except anti-apoptosis, can also mitogenesis anti-fibrosis.Except expressing relevant cell factor, MSC also expresses the factor relevant to bone forming, as skeletonization specific factor-2 (OSF-2) and BMP-1; In addition, also express nerve cell adhesion molecule neuropilin, NGF and neurotrophic factor derived from brain (BDNF), these factors can participate in the neurotization of central nervous system, promote the existence of injured brain tissue and reinvent.In addition, MSC can secrete some adhering substrate molecules (fibronectin, ln etc.) and can promote the adhesion, adherent of cell in the process of propagation.
MSC has been widely used in the clinical illness such as graft-vs-host reaction, heart failure, osteogenesis imperfecta, Spinal injury, late period dilated cardiomyopathy, richets, apoplexy, autoimmune disorder at present, and has obtained satisfied clinical effectiveness.For example, but the quantity of MSC in tissue is extremely small, only accounts for 0.001%～0.01% in medullary cell.From tissue, separate the stem cell that obtains very limited, so use the serum free medium etc. of foetal calf serum substratum, human serum substratum, people's platelet rich plasma, human blood platelets lysate and specific chemical components to carry out a large amount of amplification in vitros to meet Experiment and clinic presentations research experiment and clinical study people at present.
But at present no matter people are at experimental study, or GMP only pays close attention to the production of MSC in producing, and it is discarded that the supernatant liquor that MSC is cultivated is used as biologic garbage, and this has greatly wasted the biologically active substance that MSC secretes in culturing process.And current in the clinical treatment of MSC, the use of serum free medium has increased the cost of MSC treatment greatly, so if the supernatant liquor that MSC can be cultivated be recycled again, will have larger value so.
Summary of the invention
The object of the present invention is to provide a kind of substratum and the preparation and application thereof that can support various sources MSC in-vitro separation, cultivation, amplification use, substratum of the present invention simultaneously can backer's umbilical vein endothelial progenitor cell (HUVEC) and normal liver cell LO
2growth.
The present invention utilizes stem cell to have the feature of short ex vivo expansion of stem cell at a large amount of biologically active factors that carries out secreting in vitro culture, breeding, by the MSC of the tissues such as in-vitro separation, cultivator umbilical cord, amnion, placenta and fat, collect the supernatant liquor in culturing process, carried out the ultrafiltration membrance filter of freeze thawing, low ternperature separation process, 3K and concentrated, being obtained being rich in the concentrated solution that enriches biologically active factors; Measure the contained protein concentration of enriched material, add in basic medium according to the ratio of 0.1-10 mg/mL, be prepared into the vitro culture of perfect medium for various kinds of cell.
Method of preparation and use based on stem cell secretion factor substratum provided by the invention has following beneficial effect:
Substratum prepared by method provided by the invention does not need to add FBS, various former culture that also can sustenticular cell.
The enriched material that is rich in multiple somatomedin provided by the invention can be used as the additive of cell cultures, for medical science special cells is cultivated and new cultivation additive is opened up in treatment and the qualification of medical science special cells, the various makeup such as while MSC secretory product also can be applicable to human body beauty treatment, external preparation for skin, whitening and softening skin, delay senility.
The present invention utilizes MSC in the feature of carrying out a large amount of biologically active factors of secreting in vitro culture, breeding, collect MSC culture supernatant night, purify, concentrated active factor wherein or material be again for the vitro culture of cell, this technology reuses waste cell culture supernatant, not only provide cost savings, greatly reduce the potential hazard that waste cell culture supernatant exists physical environment and human health as biologic garbage simultaneously.
Non-animal derived, people source, the recycling of serum-free MSC substratum that preparation method based on stem cell secretion factor substratum provided by the invention can use early stage, not only greatly saved that MSC is non-animal derived, people source, serum-free culture cost, also greatly reduce the cost of MSC as cell therapy simultaneously.
Brief description of the drawings
Fig. 1 is that substratum of the present invention (a) and 10% FBS substratum (b) are cultivated 7 days cellular form figure of primary MSC;
Fig. 2 is the MSC Analytical Chemical Experiment coloration result that substratum of the present invention (a) and 10% FBS substratum (b) are cultivated;
Fig. 3 shows substratum of the present invention (a) and 10% FBS substratum (b) cultivation EPC, 16 hour cell aspect graphs;
Fig. 4 is that substratum of the present invention (a) and 10% FBS substratum (b) are cultivated LO
216 hour cell aspect graphs;
Embodiment
The invention discloses a kind of method of preparation and use based on stem cell secretion biologically active factors substratum, those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of preparation and use based on stem cell secretion biologically active factors substratum of the present invention is described by preferred embodiment, related personnel obviously can change methods and applications as herein described in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
In order to make those skilled in the art understand better technical scheme of the present invention, below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1: the collection of cell cultures and supernatant liquor
The collection of cell cultures and supernatant liquor comprises the following steps:
1. the tissue such as umbilical cord, amnion, placenta and fat that gathers Healthy People, PBS washes the remained blood in blood vessel off;
2. separate and remove vascular tissue;
3. residue tissue is shredded to about 1～2mm
3tissue block, moves in 0.1% collagenase II, 37 DEG C of digestion 2h, centrifugal 10 minutes of 2,500r/min;
4. get lower sediment and use again 0.25% tryptic digestion 20min, centrifugal 10 minutes of 2,500r/min;
5. abandon supernatant, retain precipitation, blow and beat into suspension with PBS, 100 μ m strainer filterings, centrifugal 10 minutes of 2,000r/min, obtains unicellular;
6. PBS washing 2 times, with 20,000/cm
2cell density be inoculated in MSC serum free medium, put 37 DEG C, 5%CO
2under saturated humidity environment, cultivate;
7. after 2 days, remove not attached cell, change fresh culture, within every 3～4 days, change fresh culture; Collect the MSC supernatant nutrient solution of changing ,-20 DEG C of preservations;
8. in the time that Growth of Cells converges to approximately 80%, by 3,000/cm
2density go down to posterity, collect change MSC supernatant nutrient solution ,-20 DEG C of preservations.
Embodiment 2: the preparation of substratum
The layoutprocedure of substratum is as follows:
1. the MSC supernatant nutrient solution collected in MSC culturing process is melted and gathered, through low-temperature centrifugation, 3Kda centrifugal ultrafiltration, 3, centrifugal 30 minutes of 800r/min, concentratedly obtains solution and is rich in multiple biologically active factors;
2. measure the protein content 9mg/mL of the enriched material of gained, enriched material is added in basic medium IMDM and makes new perfect medium in 5% ratio, the final concentration of protein of new substratum is 0.45mg/mL.
Embodiment 3: cell cultures
From the umbilical cord of Healthy People or placenta tissue, separate and obtain cell (method is with described in embodiment 1), by the cell obtaining with 20,000/cm
2density be inoculated in respectively the present invention and prepare in substratum and control group 10%FBS blood serum medium, put 37 DEG C, 5%CO
2under environment, cultivate.
Passage: primary cell changes liquid for every 3～4 days; In the time that Growth of Cells converges to approximately 80%, with 3,000/cm
2the density cultivation of going down to posterity.
Experimental result: Fig. 1 is the cellular form figure that primary cell grows to the 7th day.Fig. 1 shows, substratum of the present invention has the ability to support the separation and Culture of primary MSC, and compared with control group, ability of cell proliferation is suitable, and cell outline is clear, and refractivity is better than cellular control unit, and cellular form is short and small fusiformis, in the shape of a spiral or radial arrangement.
Embodiment 4: Lymphocytic phenotype detects
Get the 3rd generation cell, with after 0.25% tryptic digestion, PBS washing 3 times, adjusting cell density is 1.0 × 10
6/ mL; Add 10 μ L antibody (CD34 antibody, CD29 antibody, CD44 antibody, CD90 antibody, CD14 antibody, CD19 antibody, CD34 antibody, CD45 antibody), at 4 DEG C, hatch 30 minutes, with PBS washing 1 time, centrifugal 5 minutes of 1,000r/min, with the PBS re-suspended cell of 500 μ L, flow cytometer detects, with Cell Quest software analysis, establish a pipe contrast, detected result is in table 1.
Table 1:MSC flow cytometer detection result
Result shows, the MSC positive expression CD13 of culture medium culturing of the present invention, CD29, CD44, CD90, negative CD14, CD19, CD34, the CD45 of expressing; With the comparison of control group (10%FBS) cell, the cell positive expression rate of culture medium culturing of the present invention is all higher than control group, and wherein CD13, CD44 are apparently higher than control group, and negative expression rate is all lower than 0.5%.
Embodiment 5: cytodifferentiation ability detects
MSC is at serum-free and having after 5 generations of serum free culture system, with 1 × 10
5cells/well is inoculated in 6 orifice plates that overlay I type mouse tail collagen, reaches 50% replace medium to osteogenic induction substratum while converging until cell.Osteogenic induction substratum is that DMEM substratum adds 10% foetal calf serum, 1 μ mol/L dexamethasone, 50 μ g/mL xitix, 0.01mol/L sodium β-glycerophosphate.Control group is the DMEM substratum that adds 10% foetal calf serum.Within every 3 days, change liquid, induce after 21 days, 95% ethanol is fixed, Alizarin red staining, the outer calcium apposition of micro-Microscopic observation born of the same parents.
MSC is at serum-free and having after 5 generations of serum free culture system, with 1 × 10
5cells/well is inoculated in 6 orifice plates that overlay I type mouse tail collagen, reaches 80% replace medium to fatty inducing culture while converging until cell.Fat inducing culture is that DMEM adds 10% foetal calf serum, 10 μ g/mL Regular Insulin, 1 μ mol/L dexamethasone, 0.5 mmol/L IBMX, 0.1 mmol/L INDOMETHACIN.Control group is the DMEM substratum that adds 10% foetal calf serum.Within every 3 days, change liquid, induce after 21 days, 10% paraformaldehyde is fixed, saturated oil red-O dye liquor dyeing, and micro-Microscopic observation born of the same parents intrinsic color fat drips.
MSC is at serum-free and having after 5 generations of serum free culture system, with 1 × 10
5cells/well is inoculated in the hole central authorities of 6 orifice plates that overlay I type mouse tail collagen, after 24 hours, replaces medium to chondrocyte induction substratum.Chondrocyte induction substratum is that DMEM adds 5% foetal calf serum, 50 μ g/mL xitix, ITS, 100 μ g/mL Sodium.alpha.-ketopropionates, 100 nmol/L dexamethasone, 40 μ g/mL proline(Pro), 10ng/mL TGF-β 3.Within every 3 days, change liquid, induce after 21 days, 10% paraformaldehyde is fixed, alcian blue dyeing, micro-Microscopic observation coloration result.
Fig. 2 be substratum of the present invention with control medium (10%FBS) vitro culture MSC skeletonization, become fat, become the differentiated result of cartilage.Coloration result shows that the MSC of culture medium culturing of the present invention has skeletonization, becomes fat, becomes the ability of cartilage differentiation, and skeletonization, one-tenth cartilage differentiation ability are better than the MSC that control group is cultivated compared with control group, become fat differentiation capability there is no notable difference.
Embodiment 6: human umbilical vein endothelial progenitor cell (HUVEC) is supported culture experiment
Get degrees of fusion 80% HUVEC, after 0.25% tryptic digestion, make cell suspension, cell counting, according to 5,000/cm
2density be inoculated in respectively substratum of the present invention and contrast in HUVEC growth medium (IMDM+10%FBS).Experimental result is shown in Fig. 3.
Result demonstration, substratum of the present invention can be supported the growing multiplication of HUVEC, and cell attachment growth is polygon or fusiformis, and clear-cut is as good as compared with control group.
Embodiment 7: Human normal hepatocyte LO
2support culture experiment
Get degrees of fusion 80% LO
2, after 0.25% tryptic digestion, make cell suspension, cell counting, according to 5,000/cm
2density be inoculated in respectively substratum of the present invention and contrast LO
2in growth medium (IMDM+10%FBS).Experimental result is shown in Fig. 4.
Result demonstration, substratum of the present invention can be supported LO
2growing multiplication, cell attachment growth is paving stone shape inlays, form is polygon, clear-cut is as good as compared with control group.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. the substratum based on stem cell secretion biologically active factors, its preparation method comprises: extraction stem cell, vitro culture, amplification are gone down to posterity, collecting cell culture supernatant;-20 DEG C of preservations, melt supernatant liquor mixing, low ternperature separation process, ultrafiltration and concentrated, obtain the highly purified enriched material additive that is rich in multiple biologically active factors; The preparation of perfect medium.Feature is the mescenchymal stem cell that stem cell secretion biologically active factors derives from the tissues such as people's umbilical cord, amnion, marrow, bleeding of the umbilicus, placenta and fat, and it is rich in the material such as multiple biologically active factors and adhesion, the matrix factor.
2. according to claim 1, the described preparation method based on stem cell secretion biologically active factors substratum, is characterized in that stem cell is the mescenchymal stem cell of the tissues such as the umbilical cord, amnion, marrow, bleeding of the umbilicus, placenta and the fat that derive from Healthy People.
3. according to claim 1, it is characterized in that, described Stem cells cultured in vitro, the amplification used medium that goes down to posterity is the serum free medium etc. of foetal calf serum substratum, human serum substratum, people's platelet rich plasma, human blood platelets lysate and specific chemical components.
4. according to claim 1, the cell culture supernatant of described collection is through mixing, low ternperature separation process, ultrafiltration and concentrated, the ultrafiltration membrance filter that treatment process is 3K, volume is concentrated 1/10, remove phenol red, retains biological activity material.
5. the using method of the substratum based on stem cell secretion biologically active factors, feature is that the enriched material that is rich in multiple biologically active factors that ultrafiltration and concentration is obtained is according to protein concentration, add in basic medium with the ratio of 0.1-10mg/mL, can be used for the cultivation of various kinds of cell.
6. according to claim 5, described ultrafiltration concentrate protein concentration is 1-100mg/mL.
7. according to claim 5, described basic medium is DMEM/F12, IMDM, DMEM, α-MEM, preferably IMDM.
8. according to claim 5, the whole protein concentration that described ultrafiltration concentrate adds in basic medium is 0.1-10mg/ mL, preferably 0.5-5mg/ mL.
9. according to claim 5, described cell cultures comprises primary separation and the cultivation of going down to posterity, human umbilical vein endothelial progenitor cell (HUVEC) and the Human normal hepatocyte LO of the MSC in various sources
2growth.
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| CN106074356A (en) * | 2016-08-04 | 2016-11-09 | 深圳市合康生物科技股份有限公司 | A kind of preparation method and applications of living cells essence |
| CN106256374A (en) * | 2016-08-02 | 2016-12-28 | 朱冰 | Pharmaceutical composition containing stem cell metabolite and preparation method thereof |
| CN106434530A (en) * | 2016-11-16 | 2017-02-22 | 沈阳细胞治疗工程技术研发中心有限公司 | Endothelial cell culture solution |
| CN106511381A (en) * | 2016-11-04 | 2017-03-22 | 江苏安泰生物技术有限公司 | Composition and oral spray which contain stem cell secreted factors, and preparation method thereof |
| CN106701670A (en) * | 2015-08-05 | 2017-05-24 | 朱轶 | Methods for enhancing bioactive factor secretion capacity of mesenchymal stem cells and extracting active factors in culture solution |
| CN109251885A (en) * | 2018-09-26 | 2019-01-22 | 北京添易医学研究院 | Cure the preparation method of liquid in U.S. grade placenta basal decidua stem cell |
| CN109355248A (en) * | 2018-10-30 | 2019-02-19 | 中国科学院苏州生物医学工程技术研究所 | Livestock organisations cell prepares the method for animal cell culture liquid and its application of animal cell culture liquid obtained |
| CN109536441A (en) * | 2018-11-30 | 2019-03-29 | 佛山科学技术学院 | A kind of culture medium and preparation method thereof of fatty stem cell secretion object |
| CN110507597A (en) * | 2019-08-20 | 2019-11-29 | 苏州诺普再生医学有限公司 | A kind of composition and preparation method thereof, application |
| CN110575567A (en) * | 2019-08-20 | 2019-12-17 | 苏州诺普再生医学有限公司 | scaffold composite material and preparation method and application thereof |
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| CN114369566A (en) * | 2022-01-20 | 2022-04-19 | 中国中医科学院医学实验中心 | Culture solution for promoting proliferation and angiogenesis of vascular endothelial cells |
| CN115786258A (en) * | 2023-02-01 | 2023-03-14 | 昆明时光肌生物技术有限公司 | Preparation method of cell culture medium additive |
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| CN106701670A (en) * | 2015-08-05 | 2017-05-24 | 朱轶 | Methods for enhancing bioactive factor secretion capacity of mesenchymal stem cells and extracting active factors in culture solution |
| CN106256374A (en) * | 2016-08-02 | 2016-12-28 | 朱冰 | Pharmaceutical composition containing stem cell metabolite and preparation method thereof |
| CN106074356A (en) * | 2016-08-04 | 2016-11-09 | 深圳市合康生物科技股份有限公司 | A kind of preparation method and applications of living cells essence |
| CN106511381A (en) * | 2016-11-04 | 2017-03-22 | 江苏安泰生物技术有限公司 | Composition and oral spray which contain stem cell secreted factors, and preparation method thereof |
| CN106434530B (en) * | 2016-11-16 | 2019-06-18 | 沈阳细胞治疗工程技术研发中心有限公司 | A kind of culture fluid of endothelial cell |
| CN106434530A (en) * | 2016-11-16 | 2017-02-22 | 沈阳细胞治疗工程技术研发中心有限公司 | Endothelial cell culture solution |
| CN109251885A (en) * | 2018-09-26 | 2019-01-22 | 北京添易医学研究院 | Cure the preparation method of liquid in U.S. grade placenta basal decidua stem cell |
| CN109355248A (en) * | 2018-10-30 | 2019-02-19 | 中国科学院苏州生物医学工程技术研究所 | Livestock organisations cell prepares the method for animal cell culture liquid and its application of animal cell culture liquid obtained |
| CN113015789A (en) * | 2018-11-08 | 2021-06-22 | 整合培育株式会社 | Animal cell growth promoter, medium for animal cell culture, and animal cell culture device |
| CN109536441A (en) * | 2018-11-30 | 2019-03-29 | 佛山科学技术学院 | A kind of culture medium and preparation method thereof of fatty stem cell secretion object |
| CN110507597A (en) * | 2019-08-20 | 2019-11-29 | 苏州诺普再生医学有限公司 | A kind of composition and preparation method thereof, application |
| CN110575567A (en) * | 2019-08-20 | 2019-12-17 | 苏州诺普再生医学有限公司 | scaffold composite material and preparation method and application thereof |
| CN114369566A (en) * | 2022-01-20 | 2022-04-19 | 中国中医科学院医学实验中心 | Culture solution for promoting proliferation and angiogenesis of vascular endothelial cells |
| CN115786258A (en) * | 2023-02-01 | 2023-03-14 | 昆明时光肌生物技术有限公司 | Preparation method of cell culture medium additive |
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Application publication date: 20141015 |