CN104974978B - A kind of cultural method of Endothelial cell culture base and endothelial cell - Google Patents
A kind of cultural method of Endothelial cell culture base and endothelial cell Download PDFInfo
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Abstract
The present invention provides the cultural method of a kind of Endothelial cell culture base and endothelial cell, which includes serum-free basal medium, insulin, transferrins, vitamin C, bovine serum albumin(BSA), Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, laminin, epidermal growth factor, nonessential amino acid and Metformin.Endothelial cell culture base provided by the invention can be improved the proliferative capacity of endothelial cell under the compatibility of said components.In addition, Endothelial cell culture base provided by the invention can also improve endothelial cell at vessel patency.The experimental results showed that：Endothelial cell culture base Cultured endothelial cell provided by the invention to P10 for when, cell still grows vigorous；P10 is up to 97.2% for the bis- positive expressions of endothelial cell phenotype CD309 and CD31.
Description
Technical field
The present invention relates to field of biotechnology more particularly to the culture sides of a kind of Endothelial cell culture base and endothelial cell
Method.
Background technique
Vascular endothelial cell is also known as endothelial cell, is often referred on the simple squamous for being lining in the heart, blood vessel and lymph pipe internal surface
Skin, it forms the inner wall of blood vessel.Endothelial cell is distributed across one in brain, lymph node, lung, liver, spleen etc. organ-tissue
There is the general name of the phagocyte of common feature a bit, they swallow foreign matter, bacterium, necrosis and the tissue of aging, and also participation collective exempts from
Epidemic disease activity, including：1, vessel retraction and vasodilation, to control blood pressure；2, blood coagulation (thrombosis and fibrinolysis)；
3, artery sclerosis；4, angiogenesis；5, inflammation and swelling is (such as：Edema).6, endothelial cell also controls some substances, such as white blood cell
Pass in and out blood vessel.A mostly important function of vascular endothelial cell be exactly participate in the formation of blood vessel, and numerous cardiovascular diseases,
It is all due to apoptosis of vascular endothelial cell, bad in many cases in blood vessel organic disease caused by diabetes and other reasons
Caused by being proliferated extremely or no longer.Therefore vascular endothelial cell just becomes the hot spot much studied now, these researchs and
Experiment generally requires to use a large amount of vascular endothelial cell.
In the prior art, using M199 basal medium and 10% fetal calf serum as culture medium Cultured endothelial cell.So
And the proliferative capacity of the endothelial cell of this culture medium culture is poor.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of Endothelial cell culture base, endothelial cell provided by the invention
Culture medium can be improved the proliferative capacity of endothelial cell.
The present invention provides a kind of Endothelial cell culture base, including serum-free basal medium, insulin, transferrins,
Vitamin C, bovine serum albumin(BSA), Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, layer are viscous
Even albumen, epidermal growth factor, nonessential amino acid and Metformin.
Preferably, including 3~23 μ g/mL of transferrins；0~35ng/mL of laminin 1 and bovine serum albumin(BSA) 8~
33μg/mL。
Preferably, including 0.4~1.8mg/mL of insulin；8~30 μ g/mL of vitamin C；Nonessential amino acid 33~
8~33mmol/L of 68ng/mL and Metformin.
Preferably, including 4~18ng/mL of Basic Fibroblast Growth Factor；8~30ng/mL of vascular endothelial growth factor；It is dry
6~14ng/mL of 6~28ng/mL of Porcine HGF and epidermal growth factor.
Preferably, the serum-free basal medium includes IMDM serum-free basal medium.
Preferably, including serum-free basal medium and following components：
0.5~1.5mg/mL of insulin；5~20 μ g/mL of transferrins；9~28 μ g/mL of vitamin C；Bovine serum albumin
White 10~30 μ g/mL；5~15ng/mL of Basic Fibroblast Growth Factor；10~28ng/mL of vascular endothelial growth factor；Stem cell
8~25ng/mL of growth factor；2~34ng/mL of laminin 1；8~12ng/mL of epidermal growth factor；Nonessential amino acid
10~30mmol/L of 35~65ng/mL and Metformin.
The present invention provides a kind of cultural methods of endothelial cell, include the following steps：
Cord blood is separated, tunica albuginea confluent monolayer cells are obtained；
The tunica albuginea confluent monolayer cells are inoculated in Endothelial cell culture base described in above-mentioned technical proposal, obtain P1 through culture
For endothelial cell.
Preferably, the density of the inoculation is 4.5 × 105~5.5 × 105A/mL.
Preferably, the condition of the culture is：Temperature is 35 DEG C, CO2Volumetric concentration 5%.
Preferably, the acquisition P1 for further including after endothelial cell：
The P1 is subjected to secondary culture for endothelial cell；
The number of the secondary culture is 3~10 times.
The present invention provides a kind of Endothelial cell culture base, including serum-free basal medium, insulin, transferrins,
Vitamin C, bovine serum albumin(BSA), Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, layer are viscous
Even albumen, epidermal growth factor, nonessential amino acid and Metformin.Endothelial cell culture base provided by the invention exists
It can be improved the proliferative capacity of endothelial cell under the compatibility of said components.In addition, Endothelial cell culture base provided by the invention is also
Can improve endothelial cell at vessel patency.The experimental results showed that：Endothelial cell culture base culture endothelium provided by the invention is thin
Born of the same parents to P10 for when, cell still grows vigorous；P10 is up to for the bis- positive expressions of endothelial cell phenotype CD309 and CD31
97.2%.
Detailed description of the invention
Fig. 1 is the cell growth curve figure for the Endothelial cell culture base culture that comparative example 1 provides；
Fig. 2 is flow cytometer detection figure of the P3 for endothelial cell of the culture of comparative example 1；
Fig. 3 is flow cytometer detection figure of the P10 for endothelial cell of the culture of comparative example 1；
Fig. 4 is the cell growth curve figure for the Endothelial cell culture base culture that the embodiment of the present invention 1 provides；
Fig. 5 is flow cytometer detection figure of the P3 for endothelial cell of the culture of the embodiment of the present invention 1；
Fig. 6 is flow cytometer detection figure of the P10 for endothelial cell of the culture of the embodiment of the present invention 1；
Fig. 7 is the cell growth curve figure for the Endothelial cell culture base culture that the embodiment of the present invention 2 provides；
Fig. 8 is flow cytometer detection figure of the P3 for endothelial cell of the culture of the embodiment of the present invention 2；
Fig. 9 is flow cytometer detection figure of the P10 for endothelial cell of the culture of the embodiment of the present invention 2；
Figure 10 is the cell growth curve figure for the Endothelial cell culture base culture that the embodiment of the present invention 3 provides；
Figure 11 is flow cytometer detection figure of the P3 for endothelial cell of the culture of the embodiment of the present invention 3；
Figure 12 is flow cytometer detection figure of the P10 for endothelial cell of the culture of the embodiment of the present invention 3.
Specific embodiment
The present invention provides a kind of Endothelial cell culture base, including serum-free basal medium, insulin, transferrins,
Vitamin C, bovine serum albumin(BSA), Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, layer are viscous
Even albumen, epidermal growth factor, nonessential amino acid and Metformin.
Endothelial cell culture base provided by the invention includes serum-free basal medium.The present invention is to the serum-free basis
The source of culture medium does not have special limitation, using serum free medium well known to those skilled in the art, can such as adopt
With its commercial goods.In the present invention, the serum-free basal medium is preferably IMDM basal medium, the basis DMEM/F12
One of culture medium and RPM1640 basal medium are a variety of.
Endothelial cell culture base provided by the invention includes insulin, preferably includes 0.4~1.8mg/mL of insulin, more excellent
Choosing includes 0.5~1.5mg/mL of insulin.In a specific embodiment of the present invention, the Endothelial cell culture base includes insulin
0.5mg/mL, 1.0mg/mL or 1.5mg/mL.The present invention does not have special limitation to the source of the insulin, using this field
Insulin known to technical staff can such as use its commercial goods.In a specific embodiment of the present invention, the pancreas islet
Element purchase is in Li Lai company.In the present invention, the insulin is preferably actrapid monotard.The present invention is preferably by way of recombination
Obtain insulin.In the present invention, the insulin can adjust the metabolic process of endothelial cell, can promote endothelial cell pair
The synthesis of amino acid and protein.
Endothelial cell culture base provided by the invention includes transferrins, preferably includes 3~23 μ g/mL of transferrins, more
Preferably include 5~20 μ g/mL of transferrins.In a specific embodiment of the present invention, the Endothelial cell culture base includes turning iron
5 μ g/mL of albumen, 12 μ g/mL or 20 μ g/mL.The present invention does not have special limitation to the source of the transferrins, using ability
Transferrins known to field technique personnel can such as use its commercial goods.In a specific embodiment of the present invention, described
The brand of transferrins is ProSpec brand, is bought in Amy victory Science and Technology Ltd..Those skilled in the art can also pass through
The mode of recombination well known to those skilled in the art obtains transferrins.In the present invention, the transferrins can reduce interior
The protein degradation of chrotoplast；The intake for increasing amino acid, keeps the activity of cell.
Endothelial cell culture base provided by the invention includes vitamin C, preferably includes 8~30 μ g/mL of vitamin C, more excellent
Choosing includes 9~28 μ g/mL of vitamin C.In a specific embodiment of the present invention, institute's Endothelial cell culture base includes vitamin C 9
μ g/mL, 16 μ g/mL or 22 μ g/mL.The present invention does not have special limitation to the ascorbic source, using art technology
Vitamin C known to personnel can such as use its commercial goods.In a specific embodiment of the present invention, the vitamin C
It buys in GIBCO company.In the present invention, the vitamin C is capable of the formation of inducing endothelial cell.
Endothelial cell culture base provided by the invention includes bovine serum albumin(BSA), preferably includes 8~33 μ of bovine serum albumin(BSA)
G/mL more preferably includes 10~30 μ g/mL of bovine serum albumin(BSA).In a specific embodiment of the present invention, the endothelial cell training
Feeding base includes 10 μ g/mL of bovine serum albumin(BSA), 20 μ g/mL or 30 μ g/mL.The present invention does not have the source of the bovine serum albumin(BSA)
There is special limitation, using bovine serum albumin(BSA) well known to those skilled in the art, can such as use its commercial goods.?
In specific embodiments of the present invention, the bovine serum albumin(BSA) purchase is in Amy victory Equitech Bio company.In the present invention,
The bovine serum albumin(BSA) can play the role of physiology and mechanical protection and carrier function.
Endothelial cell culture base provided by the invention includes Basic Fibroblast Growth Factor, and it is raw to preferably include basic fibroblast
Long 4~18ng/mL of the factor more preferably includes 5~15ng/mL of Basic Fibroblast Growth Factor.In specific embodiments of the present invention
In, the Endothelial cell culture base includes Basic Fibroblast Growth Factor 5ng/mL, 8ng/mL or 12ng/mL.The present invention is to institute
The source for stating Basic Fibroblast Growth Factor does not have special limitation, raw using basic fibroblast well known to those skilled in the art
The long factor can such as use its commercial goods.In a specific embodiment of the present invention, the Basic Fibroblast Growth Factor
It buys in Ji'nan University.In the present invention, the Basic Fibroblast Growth Factor has potential Angiogensis activity.
Endothelial cell culture base provided by the invention includes vascular endothelial growth factor, preferably includes vascular endothelial growth factor
8~30ng/mL of son more preferably includes 10~28ng/mL of vascular endothelial growth factor.In a specific embodiment of the present invention, institute
Stating Endothelial cell culture base includes vascular endothelial growth factor 10ng/mL, 20ng/mL or 28ng/mL.The present invention is to the blood vessel
The source of endothelial growth factors does not have special limitation, is using vascular endothelial growth factor well known to those skilled in the art
Can, it can such as use its commercial goods.In a specific embodiment of the present invention, vascular endothelial growth factor purchase in and south
University.In the present invention, the vascular endothelial growth factor is also known as vascular permeability factor, is one group of powerful and energy
Generate the cell factor of a variety of effects.It can promote endothelial cell division and increment, assemble cytoplasm calcium, and induction of vascular is raw
At.
Endothelial cell culture base provided by the invention includes stem cell factor, preferably includes stem cell factor 6
~28ng/mL more preferably includes 8~25ng/mL of stem cell factor.In a specific embodiment of the present invention, the endothelium
Cell culture medium includes stem cell factor 8ng/mL, 17ng/mL or 20ng/mL.The present invention to the stem cell growth because
The source of son does not have special limitation, using stem cell factor well known to those skilled in the art, can such as use
Its commercial goods.In a specific embodiment of the present invention, the stem cell factor purchase is in Ji'nan University.In the present invention
In, the stem cell factor can promote the growth and breeding ability of endothelial cell.
Endothelial cell culture base provided by the invention includes laminin, preferably includes 0~35ng/ of laminin 1
ML more preferably includes 2~34ng/mL of laminin 1.In a specific embodiment of the present invention, the Endothelial cell culture base
Including laminin 1 2ng/mL, 17ng/mL or 34ng/mL.It is special that the present invention does not have the source of the laminin
Limitation, using laminin well known to those skilled in the art, can such as use its commercial goods.In tool of the invention
In body embodiment, the laminin purchase is in Life company.In the present invention, the laminin can promote endothelium
Cell is adherent, migration, grows and breaks up.
Endothelial cell culture base provided by the invention includes epidermal growth factor, preferably include epidermal growth factor 6~
14ng/mL more preferably includes 8~12ng/mL of epidermal growth factor.In a specific embodiment of the present invention, the endothelial cell
Culture medium includes epidermal growth factor 8ng/mL, 10ng/mL or 12ng/mL.Source of the present invention to the epidermal growth factor
There is no special limitation, using epidermal growth factor well known to those skilled in the art, can such as use its commercial goods.
In a specific embodiment of the present invention, the epidermal growth factor purchase is in Life company.In the present invention, the epidermal growth
The factor can promote vascular endothelial cell proliferation and maintain the ability of its division growth.
Endothelial cell culture base provided by the invention includes nonessential amino acid, preferably include nonessential amino acid 33~
68ng/mL more preferably includes 35~65ng/mL of nonessential amino acid.In a specific embodiment of the present invention, the endothelial cell
Culture medium includes nonessential amino acid 35ng/mL, 50ng/mL or 65ng/mL.Source of the present invention to the nonessential amino acid
There is no special limitation, using nonessential amino acid well known to those skilled in the art, can such as use its commercial goods.
In a specific embodiment of the present invention, the nonessential amino acid purchase is in GIBCO company.In the present invention, described nonessential
Amino acid include glycine, alanine, serine, aspartic acid, glutamic acid (and its amine), proline, arginine, histidine,
One of tyrosine and cystine are a variety of.In the present invention, the nonessential amino acid can be improved proliferation efficiency, extend
Cell generation time.
Endothelial cell culture base provided by the invention includes Metformin, preferably includes Metformin 8
~33mmol/L more preferably includes 10~30mmol/L of Metformin.In a specific embodiment of the present invention, in described
Chrotoplast culture medium includes Metformin 10mmol/L, 17mmol/L or 30mmol/L.The present invention is double to the diformazan
The source of guanidine hydrochloride does not have special limitation, using Metformin well known to those skilled in the art, such as may be used
To use its commercial goods.In a specific embodiment of the present invention, the Metformin purchase is in sunshine Li Deshiization
Work Co., Ltd.In the present invention, the Metformin can greatly improve cell Proliferation efficiency, reduce cell point
Change, maintains cellular morphology and proliferative capacity.
In the present invention, the Endothelial cell culture base preferably includes serum-free basal medium and following components：
0.5~1.5mg/mL of insulin；5~20 μ g/mL of transferrins；9~28 μ g/mL of vitamin C；Bovine serum albumin
White 10~30 μ g/mL；5~15ng/mL of Basic Fibroblast Growth Factor；10~28ng/mL of vascular endothelial growth factor；Stem cell
8~25ng/mL of growth factor；2~34ng/mL of laminin 1；8~12ng/mL of epidermal growth factor；Nonessential amino acid
10~30mmol/L of 35~65ng/mL and Metformin.
In a specific embodiment of the present invention, the Endothelial cell culture base specifically includes the culture of the basis 500mL IMDM
Base；Insulin 0.5mg/mL；5 μ g/mL of transferrins；9 μ g/mL of vitamin C；10 μ g/mL of bovine serum albumin(BSA)；Alkalinity is at fibre
Tie up growth factors 5 ng/mL；Vascular endothelial growth factor 10ng/mL；Stem cell factor 8ng/mL；Laminin 1 2ng/
mL；Epidermal growth factor 8ng/mL；Nonessential amino acid 35ng/mL and Metformin 10mmol/L.
In a specific embodiment of the present invention, the Endothelial cell culture base specifically includes the culture of the basis 500mL IMDM
Base；Insulin 1.5mg/mL；20 μ g/mL of transferrins；28 μ g/mL of vitamin C；30 μ g/mL of bovine serum albumin(BSA)；Alkalinity at
Fibroblast growth factor 15ng/mL；Vascular endothelial growth factor 28ng/mL；Stem cell factor 25ng/mL；Laminin
34ng/mL；Epidermal growth factor 12ng/mL；Nonessential amino acid 65ng/mL and Metformin 30mmol/L.
In a specific embodiment of the present invention, the Endothelial cell culture base specifically includes the culture of the basis 500mL IMDM
Base；Insulin 1mg/mL；12 μ g/mL of transferrins；16 μ g/mL of vitamin C；20 μ g/mL of bovine serum albumin(BSA)；Alkalinity is at fibre
Tie up growth factor 8ng/mL；Vascular endothelial growth factor 20ng/mL；Stem cell factor 17ng/mL；Laminin
24ng/mL；Epidermal growth factor 10ng/mL；Nonessential amino acid 50ng/mL and Metformin 17mmol/L.
In the present invention, the preparation method of the Endothelial cell culture base preferably includes following steps：
Serum-free basal medium, insulin, transferrins, vitamin C, bovine serum albumin(BSA), basic fibroblast is raw
The long factor, vascular endothelial growth factor, stem cell factor, laminin, epidermal growth factor, nonessential amino acid and
Metformin mixing, obtains Endothelial cell culture base.
In the present invention, the serum-free basal medium, insulin, transferrins, vitamin C, bovine serum albumin(BSA),
It is Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, laminin, epidermal growth factor, non-
Serum-free basal medium, pancreas islet described in the source and dosage and above-mentioned technical proposal of necessary amino acid and Metformin
Element, transferrins, vitamin C, bovine serum albumin(BSA), Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell are raw
The long factor, laminin, epidermal growth factor, nonessential amino acid and the source of Metformin are consistent with dosage,
Details are not described herein.
The present invention also provides a kind of cultural methods of endothelial cell, include the following steps：
Cord blood is separated, tunica albuginea confluent monolayer cells are obtained；
The tunica albuginea confluent monolayer cells are inoculated in Endothelial cell culture base described in above-mentioned technical proposal, obtain P1 through culture
For endothelial cell.
The present invention separates Cord blood, obtains tunica albuginea confluent monolayer cells.The present invention divides again after preferably first diluting Cord blood
From obtaining tunica albuginea confluent monolayer cells.Present invention preferably employs normal saline dilution Cord bloods.In the present invention, the physiological saline and
The volume ratio of Cord blood is preferably 1：1.Present invention preferably employs lymph separating liquids well known to those skilled in the art to Cord blood
It is separated, obtains tunica albuginea confluent monolayer cells.In the present invention, the lymph separating liquid and the volume ratio of Cord blood are preferably 6:4.5
~5.5, more preferably 6:5.In the present invention, isolated revolving speed is preferably 680g~720g, more preferably 700g；When isolated
Between preferably 15~25min, more preferably 20min.
After obtaining tunica albuginea layer, the tunica albuginea layer is inoculated in Endothelial cell culture base described in above-mentioned technical proposal by the present invention
In, P1 is obtained for endothelial cell through culture.
The present invention preferably mixes tunica albuginea layer and physiological saline, discards supernatant liquid, obtained sedimentation cell is inoculated in
It states in Endothelial cell culture base.In the present invention, the density of the inoculation is preferably 4.5 × 105~5.5 × 105A/mL.Inoculation
Afterwards, the present invention is preferably 35 DEG C in temperature, CO2The condition for the culture that volumetric concentration is 5% is cultivated.The present invention preferably cultivates
After 24 hours, liquid is discarded supernatant, rejoins the Endothelial cell culture base for discarding supernatant liquid equal volume.The present invention is preferably to thin
When born of the same parents' degrees of fusion reaches 75%~85%, liquid is discarded supernatant again, then uses trypsin digestion, it is thin to be eventually adding above-mentioned endothelium
Born of the same parents' culture medium terminates digestion.The present invention waits for that cell fusion degree reaches 75%~85%, after discarding supernatant liquid, remaining cell is preferred
Using phosphate buffer PBS clean, then with trypsase mixture slaking.Present invention preferably employs contain ethylenediamine tetra-acetic acid
(EDTA) trypsase is digested；In a particular embodiment, the trypsase containing ethylenediamine tetra-acetic acid (EDTA) is
0.25% trypsase containing 0.04%EDTA；The additional amount of the trypsase is preferably 1mL/40cm2Culture bottle bottom
Area.In the present invention, the volume ratio for terminating the Endothelial cell culture base and trypsase that digest is preferably 4~6:1, more
Preferably 5:1.
The present invention will preferably terminate postdigestive cell centrifugation, be inoculated with again, culture obtains P1 for endothelial cell.The present invention
It is preferred that will terminate postdigestive cell is centrifuged 4~6min at 380g~420g.In the present invention, the density being inoculated with again
Preferably 4.5 × 105~5.5 × 105A/mL.After being inoculated with again, the present invention is preferably 35 DEG C in temperature, CO2Volumetric concentration is
The condition of 5% culture is cultivated, and obtains P1 for endothelial cell.
After P1 is obtained for endothelial cell, the P1 is preferably carried out secondary culture for cell by the present invention.The present invention is to passage
Method there is no special limitation, using passaging techniques scheme well known to those skilled in the art.The present invention preferably repeats
To the step of P1 is for endothelial cell is obtained after the inoculation of tunica albuginea confluent monolayer cells, the P1 is subjected to secondary culture for endothelial cell.At this
In invention, the P1 is preferably 3~10 times for the number of endothelial cell secondary culture.
The present invention carries out the surface antigen characteristic of endothelial cell using flow cytometer Human Umbilical Vein Endothelial Cells.In the present invention,
The endothelial cell preferably uses the surface antigen of CD309 antibody and CD31 antibody to detect.
The present invention provides a kind of Endothelial cell culture base, including serum-free basal medium, insulin, transferrins,
Vitamin C, bovine serum albumin(BSA), Basic Fibroblast Growth Factor, vascular endothelial growth factor, stem cell factor, layer are viscous
Even albumen, epidermal growth factor, nonessential amino acid and Metformin.Endothelial cell culture base provided by the invention exists
It can be improved the proliferative capacity of endothelial cell under the compatibility of said components.In addition, Endothelial cell culture base provided by the invention is also
Can improve endothelial cell at vessel patency.The experimental results showed that：Endothelial cell culture base culture endothelium provided by the invention is thin
Born of the same parents to P10 for when, cell still grows vigorous；P10 is up to for the bis- positive expressions of endothelial cell phenotype CD309 and CD31
97.2%.
Any animal blood serum is not contained in Endothelial cell culture base provided by the invention, will not bring any mycoplasma and disease into
Poison avoids security risks that may be present.
In order to further illustrate the present invention, below with reference to embodiment to a kind of Endothelial cell culture base provided by the invention and
The cultural method of endothelial cell is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Comparative example 1
By M199 basal medium (purchase is in GIBCO)+10% fetal calf serum (purchase is in GIBCO) mixing, cultivated
Base.
Step 1：By the Cord blood of fresh acquisition normal saline dilution 1:1 dilution, mixes；
Step 2：50mL centrifuge tube is taken, 15mL FICO lymphocyte separation medium is added, the navel after being subsequently added into 25mL dilution
Band blood, 700g are centrifuged 20 minutes；
Step 3：Tunica albuginea layer is drawn, 30mL physiological saline is added and mixes, 500g is centrifuged 5 minutes, is discarded supernatant, and precipitating is used
Culture medium is resuspended, according to 5 × 105Cell is resuspended in the density of/mL, and cell suspension is added in culture bottle and is put in 37 DEG C, 5%CO2's
It is cultivated in incubator；
Step 4：Liquid is discarded supernatant after 24 hours, rejoins the above-mentioned culture medium of supernatant volume；
Step 5：When cell fusion degree reaches 80%, liquid is discarded supernatant, is cleaned 2 times with PBS, addition contains 0.04%
0.25% trypsin digestion of EDTA, pancreatin additional amount are 1mL/40cm2Culture bottle floor space adds after cell detachment
The culture medium for entering 5 times of pancreatin volumes terminates digestion；
Step 6：Cell 400g under digestion is centrifuged 5 minutes, liquid is discarded supernatant, by cell according to 5 × 105/ mL's is close
Degree is resuspended with above-mentioned culture medium, is inoculated into culture bottle and is put into 37 DEG C, 5%CO2Incubator in cultivate, which is denoted as P1
Generation；
Step 5, step 6 are repeated by cell culture to P10 generation.
The endothelial cell number for the culture medium culture that 1 comparative example 1 of table provides
Fig. 1 is the cell growth curve figure for the Endothelial cell culture base culture that comparative example 1 provides.
Fig. 2 is flow cytometer detection figure of the P3 for endothelial cell of the culture of comparative example 1.From figure 2 it can be seen that P3 is for cell
CD309+CD31+ expression rate is 35.7%.
Fig. 3 is flow cytometer detection figure of the P10 for endothelial cell of the culture of comparative example 1.From figure 3, it can be seen that P10 is for cell
CD309+CD31+ expression rate be 0%.
Embodiment 1
By 250mg rh-insulin, 2.5mg recombinant transferrin, 4.5mg vitamin C, 5g bovine serum albumin(BSA), 2.5
μ g Basic Fibroblast Growth Factor, 5 μ g vascular endothelial growth factor, 4 μ g stem cell factors, 6 μ g laminins, 4 μ g
Epidermal growth factor, 17.5g nonessential amino acid, 828.15mg Metformin and 500mL IMDM basal medium are mixed
It closes, obtains Endothelial cell culture base.
Step 1：By Cord blood normal saline dilution 1:1 dilution, mixes；
Step 2：50mL centrifuge tube is taken, 15mL FICOLL lymphocyte separation medium is added, after being subsequently added into 25mL dilution
Cord blood, 700g are centrifuged 20 minutes；
Step 3：Tunica albuginea layer is drawn, 30mL physiological saline is added and mixes, 500g is centrifuged 5 minutes, is discarded supernatant liquid, will be precipitated
It is outstanding with above-mentioned Endothelial cell culture base weight, according to 5 × 105Cell is resuspended in the density of/mL, and cell suspension is added in culture bottle and is put
In 37 DEG C, the CO that volumetric concentration is 5%2Incubator in cultivate；
Step 4：Liquid is discarded supernatant after 24 hours, rejoins the above-mentioned Endothelial cell culture base of supernatant volume；
Step 5：When cell fusion degree reaches 80%, liquid is discarded supernatant, is cleaned 2 times with PBS, addition contains 0.04%
0.25% trypsin digestion of EDTA, trypsase additional amount are 1mL/40cm2Culture bottle floor space.To cell detachment
Afterwards, the above-mentioned Endothelial cell culture base that 5 times of trypsase volumes are added terminates digestion；
Step 6：It will be centrifuged 5 minutes under cell 400g under digestion, liquid discarded supernatant, by cell according to 5 × 105/ mL's
Density is outstanding with above-mentioned Endothelial cell culture base weight, is inoculated into and is put into 37 DEG C in culture bottle, the CO that volumetric concentration is 5%2Culture
It is cultivated in case, which is denoted as P1 generation；
Step 5, step 6 are repeated by cell culture to P10 generation.
The endothelial cell number for the culture medium culture that 2 embodiment of the present invention 1 of table provides
Fig. 4 is the cell growth curve figure for the Endothelial cell culture base culture that the embodiment of the present invention 1 provides.It can be with from Fig. 4
Find out, culture of the present invention to P10 for when, cell growth it is still vigorous, and comparative example 1 culture to P10 for when hardly increased
It grows.
Fig. 5 is flow cytometer detection figure of the P3 for endothelial cell of the culture of the embodiment of the present invention 1.From figure 5 it can be seen that P3 generation
The CD309+CD31+ expression rate of cell is 99.5%.
Fig. 6 is flow cytometer detection figure of the P10 for endothelial cell of the culture of the embodiment of the present invention 1.From fig. 6 it can be seen that P10
CD309+CD31+ expression rate for cell is 84.5%.
The present invention test at blood vessel to the endothelial cell of culture：
By 4 DEG C of dissolutions overnight of Matrigel matrigel (being purchased from GIBCO)；
Next day carries out paving glue on ice chest, first uses physiological saline 1:1 dilution Matrigel matrigel, after mixing, 50 holes μ L/
96 orifice plates are added, 37 DEG C of incubators place 30min；
The vascular endothelial cell turned out in Example 1 and comparative example 1, is inoculated in 96 orifice plates, and every kind of cell is pressed
It is divided into 2 groups according to 10000 cell number, 4 holes of every group of inoculation are inoculated with 8 holes in total, are then placed in incubator and cultivate；
Every group is calculated after 6 hours, 12 hours, 24 hours in 100 times of amplification factors, the number of blood vessel in 10 visuals field
Amount the results are shown in Table 3, and table 3 is the endothelial cell of the embodiment of the present invention 1 and the culture of comparative example 1 into blood vessel number：
The endothelial cell that 3 embodiment of the present invention 1 of table and comparative example 1 are cultivated at 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 rh-insulin, 600 μ g recombinant transferrins, 8mg vitamin C, 5g bovine serum albumin(BSA), 4 μ g alkali
Property fibroblast growth factor, 10 μ g vascular endothelial growth factor, 8.5 μ g stem cell factors, 12 μ g laminins, 5 μ g
Epidermal growth factor, 25g nonessential amino acid, 1407.855mg Metformin and 500mL IMDM basal medium are mixed
It closes, obtains Endothelial cell culture base；
Step 1, by the Cord blood of fresh acquisition normal saline dilution 1:1 dilution, mixes；
Step 2：50mL centrifuge tube is taken, 15mL FICOLL lymphocyte separation medium is added, after being subsequently added into 25mL dilution
Cord blood, 700g are centrifuged 20 minutes；
Step 3：The tunica albuginea layer that aspiration step 2 obtains is added 30mL physiological saline and mixes, and 500g is centrifuged 5 minutes, is discarded
Clear liquid precipitating is resuspended with culture medium, according to 5 × 105Cell is resuspended in the density of/mL, and cell suspension is added in culture bottle and is put
In 37 DEG C, the CO that volumetric concentration is 5%2Incubator in cultivate；
Step 4：Liquid is discarded supernatant after 24 hours, rejoins the above-mentioned culture medium of supernatant volume；
Step 5：When cell fusion degree reaches 80%, liquid is discarded supernatant, is cleaned 2 times with PBS, addition contains 0.04%
0.25% trypsin digestion of EDTA, trypsase additional amount are 1mL/40cm2Culture bottle floor space.To cell detachment
Afterwards, the culture medium that 5 times of trypsase volumes are added terminates digestion.
Step 6：Cell 400g under digestion is centrifuged 5 minutes, liquid is discarded supernatant, by cell according to 5 × 105/ mL's is close
Degree is resuspended with culture medium 2, is inoculated into culture bottle and is put into 37 DEG C, 5%CO2Incubator in cultivate, which is denoted as P1 generation；
Step 5, step 6 are repeated by cell culture to P10 generation.
The endothelial cell number for the culture medium culture that 4 embodiment of the present invention 2 of table provides
Fig. 7 is the cell growth curve figure for the Endothelial cell culture base culture that the embodiment of the present invention 2 provides.It can be with from Fig. 7
Find out, culture of the present invention to P10 for when, cell growth it is still vigorous, and comparative example 1 culture to P10 for when hardly increased
It grows.
Fig. 8 is flow cytometer detection figure of the P3 for endothelial cell of the culture of the embodiment of the present invention 2.As can be seen from Figure 8, P3 generation
The CD309+CD31+ expression rate of cell is 99.5%.
Fig. 9 is flow cytometer detection figure of the P10 for endothelial cell of the culture of the embodiment of the present invention 2.It can be seen in figure 9 that P10
CD309+CD31+ expression rate for cell is 97.2%.
Every group is calculated after 6 hours, 12 hours, 24 hours in 100 times of amplification factors, the number of blood vessel in 10 visuals field
Amount the results are shown in Table 5, and table 5 is the endothelial cell of the embodiment of the present invention 2 and the culture of comparative example 1 into blood vessel number：
The endothelial cell that 5 embodiment of the present invention 2 of table and comparative example 1 are cultivated at 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 rh-insulin, 10mg recombinant transferrin, 14mg vitamin C, 15g bovine serum albumin(BSA), 7.5 μ
G Basic Fibroblast Growth Factor, 14 μ g vascular endothelial growth factor, 12.5 μ g stem cell factors, 17 μ g laminins,
6 μ g epidermal growth factor, 32.5g nonessential amino acid, 2484.45mg Metformin and the culture of the basis 500mL IMDM
Base mixing, obtains Endothelial cell culture base.
Step 1：By the Cord blood of fresh acquisition normal saline dilution 1:1 dilution, mixes；
Step 2：50mL centrifuge tube is taken, 15mL FICOLL lymphocyte separation medium is added, after being subsequently added into 25mL dilution
Cord blood, 700g are centrifuged 20 minutes；
Step 3：Tunica albuginea layer is drawn, 30mL physiological saline is added and mixes, 500g is centrifuged 5 minutes, is discarded supernatant liquid, will be precipitated
It is resuspended with above-mentioned culture medium, according to 5 × 105Cell is resuspended in the density of/mL, cell suspension is added in culture bottle be put in 37 DEG C,
The CO that volumetric concentration is 5%2Incubator in cultivate；
Step 4：Liquid is discarded supernatant after 24 hours, rejoins the culture medium of supernatant volume；
Step 5：When cell fusion degree reaches 80%, liquid is discarded supernatant, is cleaned 2 times with PBS, addition contains 0.04%
0.25% trypsin digestion of EDTA, trypsase additional amount are 1mL/40cm2Culture bottle floor space.To cell detachment
Afterwards, the above-mentioned culture medium that 5 times of trypsase volumes are added terminates digestion；
Step 6：Cell 400g under digestion is centrifuged 5 minutes, liquid is discarded supernatant, by cell according to 5 × 105/ mL's is close
Degree is resuspended with culture medium, is inoculated into culture bottle and is put into 37 DEG C, 5%CO2Incubator in cultivate, which is denoted as P1 generation；
Step 5, step 6 are repeated by cell culture to P10 generation.
The endothelial cell number for the culture medium culture that 6 embodiment of the present invention 3 of table provides
Figure 10 is the cell growth curve figure for the Endothelial cell culture base culture that the embodiment of the present invention 3 provides.It can from Figure 10
To find out, culture of the present invention to P10 for when, cell growth is still vigorous, and the culture of comparative example 1 to P10 for when hardly
It is proliferated.
Figure 11 is flow cytometer detection figure of the P3 for endothelial cell of the culture of the embodiment of the present invention 3.It can be seen from fig. 11 that P3
CD309+CD31+ expression rate for cell is 99.9%.
Figure 12 is flow cytometer detection figure of the P10 for endothelial cell of the culture of the embodiment of the present invention 3.In figure 12 it can be seen that
P10 is 85.0% for the CD309+CD31+ expression rate of cell.
Every group is calculated after 6 hours, 12 hours, 24 hours in 100 times of amplification factors, the number of blood vessel in 10 visuals field
Amount the results are shown in Table 7, and table 7 is the endothelial cell of the embodiment of the present invention 3 and the culture of comparative example 1 into blood vessel number：
The endothelial cell that 7 embodiment of the present invention 3 of table and comparative example 1 are cultivated at 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 present invention provides a kind of Endothelial cell culture base, including serum-free basal medium,
Insulin, vitamin C, bovine serum albumin(BSA), Basic Fibroblast Growth Factor, vascular endothelial growth factor, is done carefully transferrins
The intracellular growth factor, laminin, epidermal growth factor, nonessential amino acid and Metformin.It is provided by the invention
Endothelial cell culture base can be improved the proliferative capacity of endothelial cell under the compatibility of said components.In addition, provided by the invention
Endothelial cell culture base can also improve endothelial cell at vessel patency.The experimental results showed that：Endothelial cell provided by the invention
Culture medium Cultured endothelial cell to P10 for when, cell still grows vigorous；P10 is for the bis- sun of endothelial cell phenotype CD309 and CD31
Property expression up to 97.2%.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (6)
1. a kind of Endothelial cell culture base, composed of the following components：
Serum-free basal medium, 0.4~1.8mg/mL of insulin, 3~23 μ g/mL of transferrins, 8~30 μ g/ of vitamin C
ML, 8~33 μ g/mL of bovine serum albumin(BSA), 4~18ng/mL of Basic Fibroblast Growth Factor, vascular endothelial growth factor 8~
30ng/mL, 6~28ng/mL of stem cell factor, 0~35ng/mL of laminin 1,6~14ng/ of epidermal growth factor
8~33mmol/L of mL, 33~68ng/mL of nonessential amino acid and Metformin；The serum-free basal medium is
IMDM serum-free basal medium.
2. Endothelial cell culture base according to claim 1, which is characterized in that by serum-free basal medium and with the following group
It is grouped as：
0.5~1.5mg/mL of insulin, 5~20 μ g/mL of transferrins, 9~28 μ g/mL of vitamin C, bovine serum albumin(BSA)
10~30 μ g/mL, it 5~15ng/mL of Basic Fibroblast Growth Factor, 10~28ng/mL of vascular endothelial growth factor, does carefully
8~25ng/mL of the intracellular growth factor, 2~34ng/mL of laminin 1,8~12ng/mL of epidermal growth factor, nonessential ammonia
10~30mmol/L of 35~65ng/mL of base acid and Metformin.
3. a kind of cultural method of endothelial cell, includes the following steps：
Cord blood is separated, tunica albuginea confluent monolayer cells are obtained；
The tunica albuginea confluent monolayer cells are inoculated in Endothelial cell culture base described in claim 1~2 any one, are obtained through culture
P1 is obtained for endothelial cell.
4. cultural method according to claim 3, which is characterized in that the density of the inoculation is 4.5 × 105~5.5 ×
105A/mL.
5. cultural method according to claim 3, which is characterized in that the condition of the culture is：Temperature is 35 DEG C, CO2Body
Product concentration is 5%.
6. cultural method according to claim 3, which is characterized in that the acquisition P1 for further including after endothelial cell：
The P1 is subjected to secondary culture for endothelial cell；
The number of the secondary culture is 3~10 times.
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