CN104974978A - Endothelial cell culture medium and endothelial cell culture method - Google Patents
Endothelial cell culture medium and endothelial cell culture method Download PDFInfo
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Abstract
The invention provides an endothelial cell culture medium and an endothelial cell culture method, wherein the endothelial cell culture medium comprises a serum-free basal culture medium, insulin, transferin, vitamin C, bovine serum albumin, a basic fibroblast growth factor, a blood vessel endothelium growth factor, a stem cell growth factor, laminin, an epidermal growth factor, non-essential amino acid and melbinum hydrochloride. By virtue of compatibility of ingredients, the endothelial cell culture medium provided by the invention is capable of improving the endothelial cell multiplication capacity. In addition, the endothelial cell culture medium provided by the invention is further capable of improving the blood vessel formation capability of endothelial cells. The experimental result indicates that when endothelial cells are cultured to the P10 generation by virtue of the endothelial cell culture medium provided by the invention, the cells still grow vigorously; the P10-generation endothelial cell phenotype CD309 and CD31 double-positive expression is as high as 97.2%.
Description
Technical field
The present invention relates to biological technical field, particularly relate to the cultural method of a kind of Endothelial cell culture base and endotheliocyte.
Background technology
Vascular endothelial cell, also known as endotheliocyte, is often referred to the simple squamous epithelium being lining in the heart, blood vessel and intralymphatic surface, and its forms the inwall of blood vessel.Endotheliocyte is the cytophagous general name that some being distributed in brain, lymphoglandula, lung, liver, spleen etc. organ-tissue have common feature, they engulf foreign matter, bacterium, necrosis and old and feeble tissue, also participate in collective's Immunization Activities, comprise: 1, vasoconstriction and vasorelaxation, thus control blood pressure; 2, blood coagulation (thrombosis and fibrinolysis); 3, arteriosclerosis; 4, vasculogenesis; 5, inflammation and swelling (as: edema).6, endotheliocyte also controls some materials, as white cell turnover blood vessel.The of paramount importance function of vascular endothelial cell is exactly the formation participating in blood vessel, and in the blood vessel organic disease caused at numerous cardiovascular disorder, diabetes and other reasons, all due to apoptosis of vascular endothelial cell, necrosis or no longer propagation causes in a lot of situation.Therefore vascular endothelial cell just becomes the focus of a lot of research now, and these researchs and experiment often need to use a large amount of vascular endothelial cells.
In prior art, adopt M199 basic medium and 10% foetal calf serum as culture medium culturing endotheliocyte.But the multiplication capacity of the endotheliocyte of this culture medium culturing is poor.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of Endothelial cell culture base, Endothelial cell culture base provided by the invention can improve the multiplication capacity of endotheliocyte.
The invention provides a kind of Endothelial cell culture base, comprise serum-free basic medium, Regular Insulin, Transferrins,iron complexes, vitamins C, bovine serum albumin, Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, ln, Urogastron, nonessential amino acid and Metformin.
Preferably, Transferrins,iron complexes 3 ~ 23 μ g/mL is comprised; LN1 0 ~ 35ng/mL and bovine serum albumin 8 ~ 33 μ g/mL.
Preferably, Regular Insulin 0.4 ~ 1.8mg/mL is comprised; Vitamins C 8 ~ 30 μ g/mL; Nonessential amino acid 33 ~ 68ng/mL and Metformin 8 ~ 33mmol/L.
Preferably, Basic Fibroblast Growth Factor 4 ~ 18ng/mL is comprised; Vascular endothelial growth factor 8 ~ 30ng/mL; Stem cell factor 6 ~ 28ng/mL and Urogastron 6 ~ 14ng/mL.
Preferably, described serum-free basic medium comprises IMDM serum-free basic medium.
Preferably, serum-free basic medium and following component is comprised:
Regular Insulin 0.5 ~ 1.5mg/mL; Transferrins,iron complexes 5 ~ 20 μ g/mL; Vitamins C 9 ~ 28 μ g/mL; Bovine serum albumin 10 ~ 30 μ g/mL; Basic Fibroblast Growth Factor 5 ~ 15ng/mL; Vascular endothelial growth factor 10 ~ 28ng/mL; Stem cell factor 8 ~ 25ng/mL; LN1 2 ~ 34ng/mL; Urogastron 8 ~ 12ng/mL; Nonessential amino acid 35 ~ 65ng/mL and Metformin 10 ~ 30mmol/L.
The invention provides a kind of cultural method of endotheliocyte, comprise the following steps:
Cord blood is separated, obtains tunica albuginea confluent monolayer cells;
Described tunica albuginea confluent monolayer cells being inoculated in the Endothelial cell culture base described in technique scheme, obtaining P1 for endotheliocyte through cultivating.
Preferably, the density of described inoculation is 4.5 × 10
5~ 5.5 × 10
5individual/mL.
Preferably, the condition of described cultivation is: temperature is 35 DEG C, CO
2volumetric concentration 5%.
Preferably, described acquisition P1 also comprises for after endotheliocyte:
Described P1 is carried out Secondary Culture for endotheliocyte;
The number of times of described Secondary Culture is 3 ~ 10 times.
The invention provides a kind of Endothelial cell culture base, comprise serum-free basic medium, Regular Insulin, Transferrins,iron complexes, vitamins C, bovine serum albumin, Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, ln, Urogastron, nonessential amino acid and Metformin.Endothelial cell culture base provided by the invention can improve the multiplication capacity of endotheliocyte under the compatibility of said components.In addition, Endothelial cell culture base provided by the invention can also improve the one-tenth vessel patency of endotheliocyte.Experimental result shows: Endothelial cell culture base Cultured endothelial cell provided by the invention to P10 for time, cell still grows vigorous; P10 for the two positive expression of endothelial cell phenotype CD309 and CD31 up to 97.2%.
Accompanying drawing explanation
The Endothelial cell culture base cultured cells growth curve chart that Fig. 1 provides for comparative example 1;
Fig. 2 is the flow cytometer detection figure of P3 for endotheliocyte of comparative example 1 cultivation;
Fig. 3 is the flow cytometer detection figure of P10 for endotheliocyte of comparative example 1 cultivation;
The Endothelial cell culture base cultured cells growth curve chart that Fig. 4 provides for the embodiment of the present invention 1;
Fig. 5 is the flow cytometer detection figure of P3 for endotheliocyte of the embodiment of the present invention 1 cultivation;
Fig. 6 is the flow cytometer detection figure of P10 for endotheliocyte of the embodiment of the present invention 1 cultivation;
The Endothelial cell culture base cultured cells growth curve chart that Fig. 7 provides for the embodiment of the present invention 2;
Fig. 8 is the flow cytometer detection figure of P3 for endotheliocyte of the embodiment of the present invention 2 cultivation;
Fig. 9 is the flow cytometer detection figure of P10 for endotheliocyte of the embodiment of the present invention 2 cultivation;
The Endothelial cell culture base cultured cells growth curve chart that Figure 10 provides for the embodiment of the present invention 3;
Figure 11 is the flow cytometer detection figure of P3 for endotheliocyte of the embodiment of the present invention 3 cultivation;
Figure 12 is the flow cytometer detection figure of P10 for endotheliocyte of the embodiment of the present invention 3 cultivation.
Embodiment
The invention provides a kind of Endothelial cell culture base, comprise serum-free basic medium, Regular Insulin, Transferrins,iron complexes, vitamins C, bovine serum albumin, Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, ln, Urogastron, nonessential amino acid and Metformin.
Endothelial cell culture base provided by the invention comprises serum-free basic medium.The source of the present invention to described serum-free basic medium does not have special restriction, adopts serum free medium well known to those skilled in the art, as adopted its commercial goods.In the present invention, described serum-free basic medium is preferably one or more in IMDM basic medium, DMEM/F12 basic medium and RPM1640 basic medium.
Endothelial cell culture base provided by the invention comprises Regular Insulin, preferably includes Regular Insulin 0.4 ~ 1.8mg/mL, more preferably comprises Regular Insulin 0.5 ~ 1.5mg/mL.In a particular embodiment of the present invention, described Endothelial cell culture base comprises Regular Insulin 0.5mg/mL, 1.0mg/mL or 1.5mg/mL.The source of the present invention to described Regular Insulin does not have special restriction, adopts Regular Insulin well known to those skilled in the art, as adopted its commercial goods.In a particular embodiment of the present invention, described Regular Insulin is bought in Li Lai company.In the present invention, described Regular Insulin is preferably insulin human.The present invention obtains Regular Insulin preferably by the mode of restructuring.In the present invention, described Regular Insulin can regulate the metabolic process of endotheliocyte, can promote that endotheliocyte is to the synthesis of amino acid and protein.
Endothelial cell culture base provided by the invention comprises Transferrins,iron complexes, preferably includes Transferrins,iron complexes 3 ~ 23 μ g/mL, more preferably comprises Transferrins,iron complexes 5 ~ 20 μ g/mL.In a particular embodiment of the present invention, described Endothelial cell culture base comprises Transferrins,iron complexes 5 μ g/mL, 12 μ g/mL or 20 μ g/mL.The source of the present invention to described Transferrins,iron complexes does not have special restriction, adopts Transferrins,iron complexes well known to those skilled in the art, as adopted its commercial goods.In a particular embodiment of the present invention, the brand of described Transferrins,iron complexes is ProSpec brand, buys in the prompt Science and Technology Ltd. of Amy.Those skilled in the art also can obtain Transferrins,iron complexes by the mode of restructuring well known to those skilled in the art.In the present invention, described Transferrins,iron complexes can reduce the proteolytic degradation of endotheliocyte; Increase amino acid whose absorption, keep the activity of cell.
Endothelial cell culture base provided by the invention comprises vitamins C, preferably includes vitamins C 8 ~ 30 μ g/mL, more preferably comprises vitamins C 9 ~ 28 μ g/mL.In a particular embodiment of the present invention, institute's Endothelial cell culture base comprises vitamins C 9 μ g/mL, 16 μ g/mL or 22 μ g/mL.The present invention does not have special restriction to described ascorbic source, adopts vitamins C well known to those skilled in the art, as adopted its commercial goods.In a particular embodiment of the present invention, described vitamins C is bought in GIBCO company.In the present invention, described vitamins C can the formation of inducing endothelial cell.
Endothelial cell culture base provided by the invention comprises bovine serum albumin, preferably includes bovine serum albumin 8 ~ 33 μ g/mL, more preferably comprises bovine serum albumin 10 ~ 30 μ g/mL.In a particular embodiment of the present invention, described Endothelial cell culture base comprises bovine serum albumin 10 μ g/mL, 20 μ g/mL or 30 μ g/mL.The source of the present invention to described bovine serum albumin does not have special restriction, adopts bovine serum albumin well known to those skilled in the art, as adopted its commercial goods.In a particular embodiment of the present invention, described bovine serum albumin is bought in Amy prompt Equitech Bio company.In the present invention, described bovine serum albumin can play physiology and mechanical protection effect and carrier function.
Endothelial cell culture base provided by the invention comprises Basic Fibroblast Growth Factor, preferably includes Basic Fibroblast Growth Factor 4 ~ 18ng/mL, more preferably comprises Basic Fibroblast Growth Factor 5 ~ 15ng/mL.In a particular embodiment of the present invention, described Endothelial cell culture base comprises Basic Fibroblast Growth Factor 5ng/mL, 8ng/mL or 12ng/mL.The source of the present invention to described Basic Fibroblast Growth Factor does not have special restriction, adopts Basic Fibroblast Growth Factor well known to those skilled in the art, as adopted its commercial goods.In a particular embodiment of the present invention, described Basic Fibroblast Growth Factor is bought in Ji'nan University.In the present invention, described Basic Fibroblast Growth Factor has potential Angiogensis activity.
Endothelial cell culture base provided by the invention comprises vascular endothelial growth factor, preferably includes vascular endothelial growth factor 8 ~ 30ng/mL, more preferably comprises vascular endothelial growth factor 10 ~ 28ng/mL.In a particular embodiment of the present invention, described Endothelial cell culture base comprises vascular endothelial growth factor 10ng/mL, 20ng/mL or 28ng/mL.The source of the present invention to described vascular endothelial growth factor does not have special restriction, adopts vascular endothelial growth factor well known to those skilled in the art, as adopted its commercial goods.In a particular embodiment of the present invention, described vascular endothelial growth factor is bought in Ji'nan University.In the present invention, described vascular endothelial growth factor also known as vascular permeability factor, be one group powerful and the cytokine of multiple effect can be produced.It can promote endothelial cell division and increment, and tenuigenin calcium is assembled, and induction of vascular generates.
Endothelial cell culture base provided by the invention comprises stem cell factor, preferably includes stem cell factor 6 ~ 28ng/mL, more preferably comprises stem cell factor 8 ~ 25ng/mL.In a particular embodiment of the present invention, described Endothelial cell culture base comprises stem cell factor 8ng/mL, 17ng/mL or 20ng/mL.The source of the present invention to described stem cell factor does not have special restriction, adopts stem cell factor well known to those skilled in the art, as adopted its commercial goods.In a particular embodiment of the present invention, described stem cell factor is bought in Ji'nan University.In the present invention, described stem cell factor can promote the growth and breeding ability of endotheliocyte.
Endothelial cell culture base provided by the invention comprises ln, preferably includes LN1 0 ~ 35ng/mL, more preferably comprises LN1 2 ~ 34ng/mL.In a particular embodiment of the present invention, described Endothelial cell culture base comprises LN1 2ng/mL, 17ng/mL or 34ng/mL.The source of the present invention to described ln does not have special restriction, adopts ln well known to those skilled in the art, as adopted its commercial goods.In a particular embodiment of the present invention, described ln is bought in Life company.In the present invention, described ln can promote endotheliocyte adherent, migration, growth and differ entiation.
Endothelial cell culture base provided by the invention comprises Urogastron, preferably includes Urogastron 6 ~ 14ng/mL, more preferably comprises Urogastron 8 ~ 12ng/mL.In a particular embodiment of the present invention, described Endothelial cell culture base comprises Urogastron 8ng/mL, 10ng/mL or 12ng/mL.The source of the present invention to described Urogastron does not have special restriction, adopts Urogastron well known to those skilled in the art, as adopted its commercial goods.In a particular embodiment of the present invention, described Urogastron is bought in Life company.In the present invention, described Urogastron can promote vascular endothelial cell proliferation and maintain the ability of its division growth.
Endothelial cell culture base provided by the invention comprises nonessential amino acid, preferably includes nonessential amino acid 33 ~ 68ng/mL, more preferably comprises nonessential amino acid 35 ~ 65ng/mL.In a particular embodiment of the present invention, described Endothelial cell culture base comprises nonessential amino acid 35ng/mL, 50ng/mL or 65ng/mL.The present invention does not have special restriction to described nonessential amino acid whose source, adopts nonessential amino acid well known to those skilled in the art, as adopted its commercial goods.In a particular embodiment of the present invention, described nonessential amino acid is bought in GIBCO company.In the present invention, described nonessential amino acid comprises one or more in glycine, L-Ala, Serine, aspartic acid, L-glutamic acid (and amine), proline(Pro), arginine, Histidine, tyrosine and Gelucystine.In the present invention, described nonessential amino acid can improve proliferate efficiency, extends cell generation time.
Endothelial cell culture base provided by the invention comprises Metformin, preferably includes Metformin 8 ~ 33mmol/L, more preferably comprises Metformin 10 ~ 30mmol/L.In a particular embodiment of the present invention, described Endothelial cell culture base comprises Metformin 10mmol/L, 17mmol/L or 30mmol/L.The source of the present invention to described Metformin does not have special restriction, adopts Metformin well known to those skilled in the art, as adopted its commercial goods.In a particular embodiment of the present invention, described Metformin is bought in Li Deshi Chemical Co., Ltd. at sunshine.In the present invention, described Metformin can improve cell proliferation efficiency greatly, reduces cytodifferentiation, maintains cellular form and multiplication capacity.
In the present invention, described Endothelial cell culture base preferably includes serum-free basic medium and following component:
Regular Insulin 0.5 ~ 1.5mg/mL; Transferrins,iron complexes 5 ~ 20 μ g/mL; Vitamins C 9 ~ 28 μ g/mL; Bovine serum albumin 10 ~ 30 μ g/mL; Basic Fibroblast Growth Factor 5 ~ 15ng/mL; Vascular endothelial growth factor 10 ~ 28ng/mL; Stem cell factor 8 ~ 25ng/mL; LN1 2 ~ 34ng/mL; Urogastron 8 ~ 12ng/mL; Nonessential amino acid 35 ~ 65ng/mL and Metformin 10 ~ 30mmol/L.
In a particular embodiment of the present invention, described Endothelial cell culture base specifically comprises 500mL IMDM basic medium; Regular Insulin 0.5mg/mL; Transferrins,iron complexes 5 μ g/mL; Vitamins C 9 μ g/mL; Bovine serum albumin 10 μ g/mL; Basic Fibroblast Growth Factor 5ng/mL; Vascular endothelial growth factor 10ng/mL; Stem cell factor 8ng/mL; LN1 2ng/mL; Urogastron 8ng/mL; Nonessential amino acid 35ng/mL and Metformin 10mmol/L.
In a particular embodiment of the present invention, described Endothelial cell culture base specifically comprises 500mL IMDM basic medium; Regular Insulin 1.5mg/mL; Transferrins,iron complexes 20 μ g/mL; Vitamins C 28 μ g/mL; Bovine serum albumin 30 μ g/mL; Basic Fibroblast Growth Factor 15ng/mL; Vascular endothelial growth factor 28ng/mL; Stem cell factor 25ng/mL; Ln 34ng/mL; Urogastron 12ng/mL; Nonessential amino acid 65ng/mL and Metformin 30mmol/L.
In a particular embodiment of the present invention, described Endothelial cell culture base specifically comprises 500mL IMDM basic medium; Regular Insulin 1mg/mL; Transferrins,iron complexes 12 μ g/mL; Vitamins C 16 μ g/mL; Bovine serum albumin 20 μ g/mL; Basic Fibroblast Growth Factor 8ng/mL; Vascular endothelial growth factor 20ng/mL; Stem cell factor 17ng/mL; Ln 24ng/mL; Urogastron 10ng/mL; Nonessential amino acid 50ng/mL and Metformin 17mmol/L.
In the present invention, the preparation method of described Endothelial cell culture base preferably includes following steps:
Serum-free basic medium, Regular Insulin, Transferrins,iron complexes, vitamins C, bovine serum albumin, Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, ln, Urogastron, nonessential amino acid and Metformin are mixed, obtains Endothelial cell culture base.
In the present invention, described serum-free basic medium, Regular Insulin, Transferrins,iron complexes, vitamins C, bovine serum albumin, Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, ln, Urogastron, serum-free basic medium described in the source of nonessential amino acid and Metformin and consumption and technique scheme, Regular Insulin, Transferrins,iron complexes, vitamins C, bovine serum albumin, Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, ln, Urogastron, nonessential amino acid is consistent with consumption with the source of Metformin, do not repeat them here.
Present invention also offers a kind of cultural method of endotheliocyte, comprise the following steps:
Cord blood is separated, obtains tunica albuginea confluent monolayer cells;
Described tunica albuginea confluent monolayer cells being inoculated in the Endothelial cell culture base described in technique scheme, obtaining P1 for endotheliocyte through cultivating.
Cord blood is separated by the present invention, obtains tunica albuginea confluent monolayer cells.The present invention is separated after preferably first being diluted by Cord blood again, obtains tunica albuginea confluent monolayer cells.The present invention preferably adopts normal saline dilution Cord blood.In the present invention, the volume ratio of described physiological saline and Cord blood is preferably 1:1.The present invention preferably adopts lymph parting liquid well known to those skilled in the art to be separated Cord blood, obtains tunica albuginea confluent monolayer cells.In the present invention, the volume ratio of described lymph parting liquid and Cord blood is preferably 6:4.5 ~ 5.5, is more preferably 6:5.In the present invention, the rotating speed of separation is preferably 680g ~ 720g, is more preferably 700g; The time be separated is preferably 15 ~ 25min, is more preferably 20min.
After obtaining tunica albuginea layer, described tunica albuginea layer is inoculated in the Endothelial cell culture base described in technique scheme by the present invention, obtains P1 for endotheliocyte through cultivating.
The present invention is preferably by tunica albuginea layer and physiological saline mixing, and abandoning supernatant, is inoculated in the sedimentation cell obtained in above-mentioned Endothelial cell culture base.In the present invention, the density of described inoculation is preferably 4.5 × 10
5~ 5.5 × 10
5individual/mL.After inoculation, the present invention is preferably 35 DEG C in temperature, CO
2volumetric concentration is that the condition of the cultivation of 5% is cultivated.After the present invention preferably cultivates 24 hours, abandoning supernatant, rejoins the Endothelial cell culture base of abandoning supernatant equal volume.The present invention is preferably when cytogamy degree reaches 75% ~ 85%, and abandoning supernatant again, then uses tryptic digestion, finally adds above-mentioned Endothelial cell culture base and stops digestion.The present invention treats that cytogamy degree reaches 75% ~ 85%, after abandoning supernatant, is preferably adopted by remaining cell phosphate buffered saline buffer PBS to clean, then with trypsinase mixture slaking.The present invention preferably adopts the trypsinase containing ethylenediamine tetraacetic acid (EDTA) (EDTA) to digest; In a particular embodiment, the described trypsinase containing ethylenediamine tetraacetic acid (EDTA) (EDTA) be containing 0.04%EDTA 0.25% trypsinase; Described tryptic add-on is preferably 1mL/40cm
2culturing bottle floorage.In the present invention, be preferably 4 ~ 6:1 for the Endothelial cell culture base and tryptic volume ratio stopping digestion, be more preferably 5:1.
The present invention preferably will stop postdigestive cell centrifugation, again inoculate, and cultivate and obtain P1 for endotheliocyte.The present invention preferably will stop postdigestive cell centrifugal 4 ~ 6min under 380g ~ 420g.In the present invention, the described density again inoculated is preferably 4.5 × 10
5~ 5.5 × 10
5individual/mL.Again after inoculation, the present invention is preferably 35 DEG C in temperature, CO
2volumetric concentration is that the condition of the cultivation of 5% is cultivated, and obtains P1 for endotheliocyte.
Obtain P1 for after endotheliocyte, described P1 is preferably carried out Secondary Culture for cell by the present invention.The present invention does not have special restriction to the method gone down to posterity, and adopts passaging techniques scheme well known to those skilled in the art.To obtaining P1 for the step of endotheliocyte after the present invention preferably repeats the inoculation of tunica albuginea confluent monolayer cells, described P1 is carried out Secondary Culture for endotheliocyte.In the present invention, described P1 is preferably 3 ~ 10 times for the number of times of endotheliocyte Secondary Culture.
The present invention adopts flow cytometer Human Umbilical Vein Endothelial Cells to carry out the surface antigen characteristic of endotheliocyte.In the present invention, described endotheliocyte preferably adopts the surface antigen of CD309 antibody and CD31 antibody to detect.
The invention provides a kind of Endothelial cell culture base, comprise serum-free basic medium, Regular Insulin, Transferrins,iron complexes, vitamins C, bovine serum albumin, Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, ln, Urogastron, nonessential amino acid and Metformin.Endothelial cell culture base provided by the invention can improve the multiplication capacity of endotheliocyte under the compatibility of said components.In addition, Endothelial cell culture base provided by the invention can also improve the one-tenth vessel patency of endotheliocyte.Experimental result shows: Endothelial cell culture base Cultured endothelial cell provided by the invention to P10 for time, cell still grows vigorous; P10 for the two positive expression of endothelial cell phenotype CD309 and CD31 up to 97.2%.
Not containing any animal serum in Endothelial cell culture base provided by the invention, any mycoplasma and virus can not be brought into, avoid the security risks that may exist.
In order to further illustrate the present invention, being described in detail below in conjunction with the cultural method of embodiment to a kind of Endothelial cell culture base provided by the invention and endotheliocyte, but they can not being interpreted as limiting the scope of the present invention.
Comparative example 1
M199 basic medium (buying in GIBCO)+10% foetal calf serum (buying in GIBCO) is mixed, obtains substratum.
Step 1: the Cord blood normal saline dilution 1:1 of fresh collection is diluted, mixing;
Step 2: get 50mL centrifuge tube, adds 15mL FICO lymphocyte separation medium, then adds the Cord blood after 25mL dilution, centrifugal 20 minutes of 700g;
Step 3: draw tunica albuginea layer, add the mixing of 30mL physiological saline, centrifugal 5 minutes of 500g, supernatant discarded, by resuspended for precipitation substratum, according to 5 × 10
5the density re-suspended cell of/mL, adds cell suspension in culturing bottle and is put in 37 DEG C, 5%CO
2incubator in cultivate;
Abandoning supernatant after step 4:24 hour, rejoins the above-mentioned substratum of supernatant volume;
Step 5: when cytogamy degree reaches 80%, abandoning supernatant, cleans 2 times with PBS, add containing 0.04%EDTA 0.25% tryptic digestion, pancreatin add-on is 1mL/40cm
2culturing bottle floorage, after cell detachment, adds 5 times of substratum to pancreatin volume and stops digestion;
Step 6: by centrifugal 5 minutes of cell 400g under digestion, abandoning supernatant, by cell according to 5 × 10
5the density of/mL is resuspended with above-mentioned substratum, is inoculated in culturing bottle and puts into 37 DEG C, 5%CO
2incubator in cultivate, this cell is designated as P1 generation;
Repeating step 5, step 6 by cell cultures to P10 generation.
The endotheliocyte number of the culture medium culturing that table 1 comparative example 1 provides
The Endothelial cell culture base cultured cells growth curve chart that Fig. 1 provides for comparative example 1.
Fig. 2 is the flow cytometer detection figure of P3 for endotheliocyte of comparative example 1 cultivation.As can be seen from Figure 2, P3 is 35.7% for the CD309+CD31+ expression rate of cell.
Fig. 3 is the flow cytometer detection figure of P10 for endotheliocyte of comparative example 1 cultivation.As can be seen from Figure 3, P10 is 0% for the CD309+CD31+ expression rate of cell.
Embodiment 1
By 250mg recombinant human insulin, 2.5mg recombinant transferrin, 4.5mg vitamins C, 5g bovine serum albumin, 2.5 μ g Basic Fibroblast Growth Factors, 5 μ g vascular endothelial growth factor, 4 μ g stem cell factors, 6 μ g lns, 4 μ g Urogastrons, 17.5g non-essential amino acid, 828.15mg Metformin and the mixing of 500mL IMDM basic medium, obtain Endothelial cell culture base.
Step 1: Cord blood normal saline dilution 1:1 is diluted, mixing;
Step 2: get 50mL centrifuge tube, adds 15mL FICOLL lymphocyte separation medium, then adds the Cord blood after 25mL dilution, centrifugal 20 minutes of 700g;
Step 3: draw tunica albuginea layer, add the mixing of 30mL physiological saline, centrifugal 5 minutes of 500g, abandoning supernatant, hangs the above-mentioned Endothelial cell culture basic weight of precipitation, according to 5 × 10
5the density re-suspended cell of/mL, cell suspension is added in culturing bottle be put in 37 DEG C, volumetric concentration is the CO of 5%
2incubator in cultivate;
Abandoning supernatant after step 4:24 hour, rejoins the above-mentioned Endothelial cell culture base of supernatant volume;
Step 5: when cytogamy degree reaches 80%, abandoning supernatant, cleans 2 times with PBS, add containing 0.04%EDTA 0.25% tryptic digestion, trypsinase add-on is 1mL/40cm
2culturing bottle floorage.After cell detachment, add 5 times of above-mentioned Endothelial cell culture bases to trypsinase volume and stop digestion;
Step 6: by digestion under cell 400g under centrifugal 5 minutes, abandoning supernatant, by cell according to 5 × 10
5the density of/mL is hanged with above-mentioned Endothelial cell culture basic weight, is inoculated in culturing bottle and puts into 37 DEG C, volumetric concentration is the CO of 5%
2incubator in cultivate, this cell is designated as P1 generation;
Repeating step 5, step 6 by cell cultures to P10 generation.
The endotheliocyte number of the culture medium culturing that table 2 embodiment of the present invention 1 provides
The Endothelial cell culture base cultured cells growth curve chart that Fig. 4 provides for the embodiment of the present invention 1.As can be seen from Figure 4, the present invention cultivate P10 for time, Growth of Cells is still vigorous, and comparative example 1 cultivate to P10 for time bred hardly.
Fig. 5 is the flow cytometer detection figure of P3 for endotheliocyte of the embodiment of the present invention 1 cultivation.As can be seen from Figure 5, P3 is 99.5% for the CD309+CD31+ expression rate of cell.
Fig. 6 is the flow cytometer detection figure of P10 for endotheliocyte of the embodiment of the present invention 1 cultivation.As can be seen from Figure 6, P10 is 84.5% for the CD309+CD31+ expression rate of cell.
The present invention becomes blood vessel to test to the endotheliocyte cultivated:
Matrigel matrigel (purchased from GIBCO) 4 DEG C to be spent the night dissolving;
Next day carries out paving glue on ice chest, and first dilute Matrigel matrigel with physiological saline 1:1, after mixing, 50 μ L/ holes add 96 orifice plates, and 37 DEG C of incubators place 30min;
The vascular endothelial cell of turning out in Example 1 and comparative example 1, is inoculated in 96 orifice plates, and often kind of cell is divided into 2 groups according to the cell count of 10000, often organizes inoculation 4 holes, altogether inoculates 8 holes, then put into incubator and cultivate;
After 6 hours, 12 hours, 24 hours, calculate often that group is in 100 times of magnifications, in 10 visuals field, the quantity of blood vessel, the results are shown in Table 3, table 3 be the endotheliocyte cultivated with comparative example 1 of the embodiment of the present invention 1 become blood vessel number:
The endotheliocyte that table 3 embodiment of the present invention 1 is cultivated with comparative example 1 become blood vessel number
| 6 hourly average blood vessel numbers | 12 hourly average blood vessel numbers | 24 hourly average blood vessel numbers | |
| Embodiment 1 | 32.4 | 73.1 | 64.3 |
| Comparative example 1 | 15.8 | 28.6 | 3.2 |
Embodiment 2
By 500mg recombinant human insulin, 600 μ g recombinant transferrins, 8mg vitamins C, 5g bovine serum albumin, 4 μ g Basic Fibroblast Growth Factors, 10 μ g vascular endothelial growth factor, 8.5 μ g stem cell factors, 12 μ g lns, 5 μ g Urogastrons, 25g non-essential amino acid, 1407.855mg Metformin and the mixing of 500mL IMDM basic medium, obtain Endothelial cell culture base;
Step 1, the Cord blood normal saline dilution 1:1 of fresh collection to be diluted, mixing;
Step 2: get 50mL centrifuge tube, adds 15mL FICOLL lymphocyte separation medium, then adds the Cord blood after 25mL dilution, centrifugal 20 minutes of 700g;
Step 3: the tunica albuginea layer that aspiration step 2 obtains, add the mixing of 30mL physiological saline, centrifugal 5 minutes of 500g, abandoning supernatant, by resuspended for precipitation substratum, according to 5 × 10
5the density re-suspended cell of/mL, cell suspension is added in culturing bottle be put in 37 DEG C, volumetric concentration is the CO of 5%
2incubator in cultivate;
Abandoning supernatant after step 4:24 hour, rejoins the above-mentioned substratum of supernatant volume;
Step 5: when cytogamy degree reaches 80%, abandoning supernatant, cleans 2 times with PBS, add containing 0.04%EDTA 0.25% tryptic digestion, trypsinase add-on is 1mL/40cm
2culturing bottle floorage.After cell detachment, add 5 times of substratum to trypsinase volume and stop digestion.
Step 6: by centrifugal 5 minutes of cell 400g under digestion, abandoning supernatant, by cell according to 5 × 10
5the density substratum 2 of/mL is resuspended, is inoculated in culturing bottle and puts into 37 DEG C, 5%CO
2incubator in cultivate, this cell is designated as P1 generation;
Repeating step 5, step 6 by cell cultures to P10 generation.
The endotheliocyte number of the culture medium culturing that table 4 embodiment of the present invention 2 provides
The Endothelial cell culture base cultured cells growth curve chart that Fig. 7 provides for the embodiment of the present invention 2.As can be seen from Figure 7, the present invention cultivate P10 for time, Growth of Cells is still vigorous, and comparative example 1 cultivate to P10 for time bred hardly.
Fig. 8 is the flow cytometer detection figure of P3 for endotheliocyte of the embodiment of the present invention 2 cultivation.As can be seen from Figure 8, P3 is 99.5% for the CD309+CD31+ expression rate of cell.
Fig. 9 is the flow cytometer detection figure of P10 for endotheliocyte of the embodiment of the present invention 2 cultivation.As can be seen from Figure 9, P10 is 97.2% for the CD309+CD31+ expression rate of cell.
After 6 hours, 12 hours, 24 hours, calculate often that group is in 100 times of magnifications, in 10 visuals field, the quantity of blood vessel, the results are shown in Table 5, table 5 be the endotheliocyte cultivated with comparative example 1 of the embodiment of the present invention 2 become blood vessel number:
The endotheliocyte that table 5 embodiment of the present invention 2 is cultivated with comparative example 1 become blood vessel number
| 6 hourly average blood vessel numbers | 12 hourly average blood vessel numbers | 24 hourly average blood vessel numbers | |
| Embodiment 2 | 28 | 67 | 59 |
| Comparative example 1 | 14.5 | 23.5 | 4 |
Embodiment 3
By 750mg recombinant human insulin, 10mg recombinant transferrin, 14mg vitamins C, 15g bovine serum albumin, 7.5 μ g Basic Fibroblast Growth Factors, 14 μ g vascular endothelial growth factor, 12.5 μ g stem cell factors, 17 μ g lns, 6 μ g Urogastrons, 32.5g non-essential amino acid, 2484.45mg Metformin and the mixing of 500mL IMDM basic medium, obtain Endothelial cell culture base.
Step 1: the Cord blood normal saline dilution 1:1 of fresh collection is diluted, mixing;
Step 2: get 50mL centrifuge tube, adds 15mL FICOLL lymphocyte separation medium, then adds the Cord blood after 25mL dilution, centrifugal 20 minutes of 700g;
Step 3: draw tunica albuginea layer, adds the mixing of 30mL physiological saline, centrifugal 5 minutes of 500g, abandoning supernatant, and precipitation is resuspended with above-mentioned substratum, according to 5 × 10
5the density re-suspended cell of/mL, cell suspension is added in culturing bottle be put in 37 DEG C, volumetric concentration is the CO of 5%
2incubator in cultivate;
Abandoning supernatant after step 4:24 hour, rejoins the substratum of supernatant volume;
Step 5: when cytogamy degree reaches 80%, abandoning supernatant, cleans 2 times with PBS, add containing 0.04%EDTA 0.25% tryptic digestion, trypsinase add-on is 1mL/40cm
2culturing bottle floorage.After cell detachment, add 5 times of above-mentioned substratum to trypsinase volume and stop digestion;
Step 6: by centrifugal 5 minutes of cell 400g under digestion, abandoning supernatant, by cell according to 5 × 10
5the density substratum of/mL is resuspended, is inoculated in culturing bottle and puts into 37 DEG C, 5%CO
2incubator in cultivate, this cell is designated as P1 generation;
Repeating step 5, step 6 by cell cultures to P10 generation.
The endotheliocyte number of the culture medium culturing that table 6 embodiment of the present invention 3 provides
The Endothelial cell culture base cultured cells growth curve chart that Figure 10 provides for the embodiment of the present invention 3.As can be seen from Figure 10, the present invention cultivate P10 for time, Growth of Cells is still vigorous, and comparative example 1 cultivate to P10 for time bred hardly.
Figure 11 is the flow cytometer detection figure of P3 for endotheliocyte of the embodiment of the present invention 3 cultivation.As can be seen from Figure 11, P3 is 99.9% for the CD309+CD31+ expression rate of cell.
Figure 12 is the flow cytometer detection figure of P10 for endotheliocyte of the embodiment of the present invention 3 cultivation.As can be seen from Figure 12, P10 is 85.0% for the CD309+CD31+ expression rate of cell.
After 6 hours, 12 hours, 24 hours, calculate often that group is in 100 times of magnifications, in 10 visuals field, the quantity of blood vessel, the results are shown in Table 7, table 7 be the endotheliocyte cultivated with comparative example 1 of the embodiment of the present invention 3 become blood vessel number:
The endotheliocyte that table 7 embodiment of the present invention 3 is cultivated with comparative example 1 become blood vessel number
| 6 hourly average blood vessel numbers | 12 hourly average blood vessel numbers | 24 hourly average blood vessel numbers | |
| Embodiment 3 | 31 | 64 | 48 |
| Comparative example 1 | 18 | 21 | 2.5 |
As seen from the above embodiment, the invention provides a kind of Endothelial cell culture base, comprise serum-free basic medium, Regular Insulin, Transferrins,iron complexes, vitamins C, bovine serum albumin, Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, ln, Urogastron, nonessential amino acid and Metformin.Endothelial cell culture base provided by the invention can improve the multiplication capacity of endotheliocyte under the compatibility of said components.In addition, Endothelial cell culture base provided by the invention can also improve the one-tenth vessel patency of endotheliocyte.Experimental result shows: Endothelial cell culture base Cultured endothelial cell provided by the invention to P10 for time, cell still grows vigorous; P10 for the two positive expression of endothelial cell phenotype CD309 and CD31 up to 97.2%.
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 (10)
1. an Endothelial cell culture base, comprises serum-free basic medium, Regular Insulin, Transferrins,iron complexes, vitamins C, bovine serum albumin, Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, ln, Urogastron, nonessential amino acid and Metformin.
2. Endothelial cell culture base according to claim 1, is characterized in that, comprises Transferrins,iron complexes 3 ~ 23 μ g/mL; LN1 0 ~ 35ng/mL and bovine serum albumin 8 ~ 33 μ g/mL.
3. Endothelial cell culture base according to claim 1, is characterized in that, comprises Regular Insulin 0.4 ~ 1.8mg/mL; Vitamins C 8 ~ 30 μ g/mL; Nonessential amino acid 33 ~ 68ng/mL and Metformin 8 ~ 33mmol/L.
4. Endothelial cell culture base according to claim 1, is characterized in that, comprises Basic Fibroblast Growth Factor 4 ~ 18ng/mL; Vascular endothelial growth factor 8 ~ 30ng/mL; Stem cell factor 6 ~ 28ng/mL and Urogastron 6 ~ 14ng/mL.
5. Endothelial cell culture base according to claim 1, is characterized in that, described serum-free basic medium comprises IMDM serum-free basic medium.
6. Endothelial cell culture base according to claim 1, is characterized in that, comprises serum-free basic medium and following component:
Regular Insulin 0.5 ~ 1.5mg/mL; Transferrins,iron complexes 5 ~ 20 μ g/mL; Vitamins C 9 ~ 28 μ g/mL; Bovine serum albumin 10 ~ 30 μ g/mL; Basic Fibroblast Growth Factor 5 ~ 15ng/mL; Vascular endothelial growth factor 10 ~ 28ng/mL; Stem cell factor 8 ~ 25ng/mL; LN1 2 ~ 34ng/mL; Urogastron 8 ~ 12ng/mL; Nonessential amino acid 35 ~ 65ng/mL and Metformin 10 ~ 30mmol/L.
7. a cultural method for endotheliocyte, comprises the following steps:
Cord blood is separated, obtains tunica albuginea confluent monolayer cells;
Described tunica albuginea confluent monolayer cells being inoculated in the Endothelial cell culture base described in claim 1 ~ 6 any one, obtaining P1 for endotheliocyte through cultivating.
8. cultural method according to claim 7, is characterized in that, the density of described inoculation is 4.5 × 10
5~ 5.5 × 10
5individual/mL.
9. cultural method according to claim 7, is characterized in that, the condition of described cultivation is: temperature is 35 DEG C, CO
2volumetric concentration is 5%.
10. cultural method according to claim 7, is characterized in that, described acquisition P1 also comprises for after endotheliocyte:
Described P1 is carried out Secondary Culture for endotheliocyte;
The number of times of described Secondary Culture is 3 ~ 10 times.
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| CN105647851A (en) * | 2016-03-01 | 2016-06-08 | 赛百慷（上海）生物技术股份有限公司 | Endothelium culture system |
| 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 |
| CN109468267A (en) * | 2018-12-19 | 2019-03-15 | 广州赛莱拉干细胞科技股份有限公司 | A kind of preparation method of Endometrial stem cell |
| CN110607278A (en) * | 2019-09-26 | 2019-12-24 | 深圳市拓普生物科技有限公司 | Tumor cell culture and application thereof |
| CN112980776A (en) * | 2021-03-23 | 2021-06-18 | 上海简巨医学生物工程有限公司 | Application of niobium carbide nano material and cell culture medium for promoting cell proliferation |
| 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 |
| CN114990065A (en) * | 2022-05-16 | 2022-09-02 | 康妍葆(北京)干细胞科技有限公司 | Directional inducer of neural stem cells, induction method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105647851A (en) * | 2016-03-01 | 2016-06-08 | 赛百慷（上海）生物技术股份有限公司 | Endothelium culture system |
| 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 |
| 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 |
| CN109468267A (en) * | 2018-12-19 | 2019-03-15 | 广州赛莱拉干细胞科技股份有限公司 | A kind of preparation method of Endometrial stem cell |
| CN110607278A (en) * | 2019-09-26 | 2019-12-24 | 深圳市拓普生物科技有限公司 | Tumor cell culture and application thereof |
| CN110607278B (en) * | 2019-09-26 | 2021-06-01 | 深圳市拓普生物科技有限公司 | Tumor cell culture and application thereof |
| CN112980776A (en) * | 2021-03-23 | 2021-06-18 | 上海简巨医学生物工程有限公司 | Application of niobium carbide nano material and cell culture medium for promoting cell proliferation |
| 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 |
| CN114990065A (en) * | 2022-05-16 | 2022-09-02 | 康妍葆(北京)干细胞科技有限公司 | Directional inducer of neural stem cells, induction method and application |
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