CN103045534A - Aged rat brain vascular endothelial cell culture fluid - Google Patents
Aged rat brain vascular endothelial cell culture fluid Download PDFInfo
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- CN103045534A CN103045534A CN2013100259435A CN201310025943A CN103045534A CN 103045534 A CN103045534 A CN 103045534A CN 2013100259435 A CN2013100259435 A CN 2013100259435A CN 201310025943 A CN201310025943 A CN 201310025943A CN 103045534 A CN103045534 A CN 103045534A
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Abstract
The invention relates to an aged rat brain vascular endothelial cell culture fluid. On the basis of each 100 ml of DMEM (Dulbecco Modified Eagle Medium) culture medium, components including plasma derived serum (PDS), penicillin-streptomycin, L-glutamine, gentamicin, heparin, an alkaline fiber growth factor, an endothelial cell growth replenishing factor, vitamin C, a vascular endothelial growth factor, an insulin-like growth factor-1 and an endothelium growth factor are added. According to the aged rat brain vascular endothelial cell culture fluid, the PDS is adopted for replacing FBS (Fetal Bovine Serum), beef calf serum or horse serum in the traditional culture fluid to ensure that the purity of brain vascular endothelial cells is increased by 30 percent; and the vitamin C and puromycin are added to ensure that the brain vascular endothelial cells grow more rapidly and purely.
Description
Technical field
The present invention relates to a kind of senile rat cerebrovascular endothelial cell nutrient solution, belong to biological technical field.
Background technology
The former culture of existing cerebral vascular endothelial cells rats, source of drawing material was newborn rat (＜10 days, 35～50g), young rat (＜3weeks, 80～100g) or the 2-3 month young adult rats (200g～300g), and the cerebrovascular endothelial cell of cultivating can only go down to posterity once, can not be frozen, and utilize cerebral vascular endothelial cells rats to cause the shortcoming that the time is long, experimental cost is high as experiment material.As: Bowman PD, Betz AL, Ar D, Wolinsky JS, Penney JB, Shivers RR, Goldstein GW.1981.Primary Culture of Capillary Endothelium From Rat Brain.INVITRO.17 (4), 353-362.
Senile rat (19～22 months, the cerebrovascular endothelial cell of 900g～1200g) is cultivated successful precedent so far there are no and reports; Existing object and the culture technique of drawing materials can not satisfy the old hemato encephalic barrier model of external foundation, studies the physiological property of old hemato encephalic barrier, and the requirement of old nervous system disorders degeneration relevant with hemato encephalic barrier.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of senile rat cerebrovascular endothelial cell nutrient solution is provided.
Technical solution of the present invention is as follows:
A kind of senile rat cerebrovascular endothelial cell nutrient solution on the basis of per hundred milliliters of DMEM substratum, adds following component:
The serum in 20ml-30ml blood plasma source, 100ul penicillin-Streptomycin sulphate solution, 1.0-1.5ml L-glutaminate, 50-150 μ l sulphur fine horse gentamicin, 1.0-1.5ml heparin, 150-300 μ l alkaline fiber is given birth to the sub-factor, 1.0-1.5ml the endothelial cell growth recruitment factor, 200-400 μ l vitamins C, 100-200 μ l vascular endothelial growth factor, 100-200 μ l insulin-like growth factor-i, 100-200 μ l endothelium is given birth to the sub-factor.
Adding said components in the DMEM substratum gets final product.
Preferred according to the present invention, described on the basis of per hundred milliliters of DMEM substratum, add following component:
The serum in 20ml blood plasma source, 100ul penicillin-Streptomycin sulphate solution, 1.0ml L-glutaminate, 100 μ l sulphur fine horse gentamicins, 1.0ml heparin, 150 μ l alkaline fibers are given birth to the sub-factor, 1.0ml endothelial cell growth recruitment factor (ECGS), 300 μ l vitamins Cs, 150 μ l vascular endothelial growth factor (VEGF), 150 μ l insulin-like growth factor-is (IGF-1), 150 μ l endotheliums are given birth to the sub-factor (EGF).
Mentioned reagent is commercially available prod commonly used, this area.
Beneficial effect
1, culture fluid of endothelial cell of the present invention adopts the serum in PDS(blood plasma source) replaced FBS (foetal calf serum), calf serum (BCS) or horse serum (HS) in traditional nutrient solution, make the cerebrovascular endothelial cell purity of cultivation increase by 30%; Major cause is owing to do not contain the PDGF(platelet-derived growth factor among the FDS), and PDGF is conducive to sneak into the smooth muscle cell in the cerebrovascular endothelial cell, the division of fibrocyte and spongiocyte and propagation; Eliminated this impact with PDS.
2, the present invention has added vitamins C and tetracycline in culture fluid of endothelial cell, and this makes the cerebrovascular endothelial cell growth faster purer.
3, the present invention has also added vascular endothelial growth factor in culture fluid of endothelial cell, insulin-like growth factor-i, endothelium is given birth to the sub-factor, and these factors can make cerebrovascular endothelial cell grow faster more healthyly, and are especially even more important to the growth of senile rat cerebrovascular endothelial cell.
Description of drawings
The former culture of senile rat cerebrovascular endothelial cell that the senile rat cerebrovascular endothelial cell that Fig. 1, the present invention make passes the preparation of 3 generations and traditional method through former culture passes the column comparison diagram of the survival rate in 3 generations;
The column comparison diagram of senile rat cerebrovascular endothelial cell the survival rate through cryopreservation resuscitation after of the senile rat cerebrovascular endothelial cell that Fig. 2, the present invention make through preparing with traditional method behind the cryopreservation resuscitation;
Fig. 3: the column comparison diagram of the senile rat cerebrovascular endothelial cell cell purity of the senile rat cerebrovascular endothelial cell that the present invention makes and traditional method preparation.
Embodiment
Below in conjunction with embodiment technical scheme of the present invention is described further, but institute of the present invention protection domain is not limited to this.
Raw material sources:
The senile rat of the 19-22 month is available from Beijing dimension tonneau China laboratory animal technique center;
The source of preparation senile rat cerebrovascular endothelial cell substratum all ingredients:
The DMEM substratum, available from Gibico company, article number 11995-065;
The serum (PDS, Plasma-Derived Serum) in blood plasma source, available from Bioquote company, article number BT-214-10;
Penicillin-Streptomycin sulphate solution, available from sigma company, article number p0718;
L-glutaminate, available from Sigma company, article number G7513;
Sulphur fine horse gentamicin, available from sigma company, article number G1397;
Heparin, available from Sigma company, article number H3149;
Alkaline fiber is given birth to the sub-factor (bFGF), available from Sigma company, and article number F9786;
Endothelial cell growth recruitment factor (ECGS), available from Sigma company, article number E2759;
Vitamins C, available from Sigma company, article number c1768;
Vascular endothelial growth factor (VEGF), available from Lonza company, article number cc4176;
Insulin-like growth factor-i (IGF-1) is available from Lonza company, article number cc4176;
Endothelium is given birth to the sub-factor (EGF) available from Lonza company, article number cc4176;
Tetracycline (Puromycin), available from Sigma company, article number p8833.
The source of various enzymes and other related reagents:
Foetal calf serum (FBS) is available from Invitrogen company, article number 10437-028;
Collagenase (Collagenase) is available from Invitrogen company, article number 17101;
DNA enzyme (DNase) is available from Sigma-Aldrich company, article number D4263;
Pancreatin (Typsin) is available from Sigma-Aldrich company, article number T3053;
Bovine serum albumin (BSA) is available from Sigma-Aldrich company, article number a3059;
Collagen iv (Collagen IV) is available from Sigma-Aldrich company, article number C5533:
Fibronectin (Fibronectin) is available from Sigma-Aldrich company, article number F1141;
Percoll is available from Sigma-Aldrich company, article number 4937;
Lumbar puncture needle (Lumbar puncture needle) is available from BD company, article number 405182.
Embodiment 1
A kind of senile rat cerebrovascular endothelial cell nutrient solution on the basis of per hundred milliliters of DMEM substratum, adds following component:
The serum in 30ml blood plasma source, 100 μ l penicillin-Streptomycin sulphate solution, 1.0ml L-glutaminate, 100 μ l sulphur fine horse gentamicins, 1.5ml heparin, 150 μ l alkaline fibers are given birth to the sub-factor, 1.0ml the endothelial cell growth recruitment factor, 400 μ l vitamins Cs, 200 μ l vascular endothelial growth factor, 200 μ l insulin-like growth factor-is, 200 μ l endotheliums are given birth to the sub-factor;
Adding said components in the DMEM substratum gets final product.
Embodiment 2
A kind of senile rat cerebrovascular endothelial cell nutrient solution on the basis of per hundred milliliters of DMEM substratum, adds following component:
The serum in 20ml blood plasma source, 100 μ l penicillin-Streptomycin sulphate solution, 1.0ml L-glutaminate, 100 μ l sulphur fine horse gentamicins, 1.0ml heparin, 100 μ l alkaline fibers are given birth to the sub-factor, 1.0ml the endothelial cell growth recruitment factor, 200 μ l vitamins Cs, 100 μ l vascular endothelial growth factor, 100 μ l insulin-like growth factor-is, 100 μ l endotheliums are given birth to the sub-factor;
Adding said components in the DMEM substratum gets final product.
Comparative Examples
Tradition cerebrovascular endothelial cell nutrient solution be the following composition of basis adding at every 100ml F12 substratum (Ham ' s-F12 substratum):
10-20ml calf serum (BCS), 100 μ l penicillin-Streptomycin sulphates are molten.(this prescription is recorded in Bowman PD, and et al.Primary Culture of Capillary Endothelium From Rat Brain.INVITRO.1981.17 (4) is among the 353-362).
Or:
44ml DMEM substratum, 44ml Ham ' s-F12 substratum, 10ml foetal calf serum (FBS), 100 μ l penicillin-Streptomycin sulphates are molten, 1.0ml heparin sodium, 1.0ml endothelial cell growth recruitment factor.(this prescription is recorded in Tunkel AR et al.Blood-brain barrier alterations in bacterial meningitis:development of an in vitro model andobservations on the effects of lipopolysaccharide.In Vitro Cell Dev Biol.199127A (2): 113-20).
Test example
Above-mentioned nutrient solution is tested cultivation as follows:
(1) behind the senile rat broken end with 20 monthly ages, peel off rat head skin, then the rat head be immersed in 0 ℃ contain the 2%(percent by volume) in foetal calf serum (FBS) parting liquid 5 minutes, take out, cut two hemicerebrums, respectively the common filter paper of two brain hemisphere in sterilization is rolled a week, then remove brain stem and white matter part, keep the grey matter part, make sample tissue;
Parting liquid is on the basis of DMEM substratum, and per hundred milliliters add following component: 1ml penicillin-Streptomycin sulphate solution; 100 μ l sulphur fine horse gentamicins;
(2) sample tissue place 5ml ice-cold contain the 2%(percent by volume) culture dish of FBS parting liquid is cut into 1～2mm
3The sample tissue fritter, add the 2%(percent by volume) the FBS parting liquid is to 25ml, disperse through piping and druming, then will blow and beat the sample tissue fritter that disperses and add the collagenase of 2.5ml and the DNA enzyme solution of 250 μ l, 100rpm/min digestion is 90 minutes in 36 ℃ shaking bath; Then after piping and druming disperses, continue digestion 30 minutes, make cell dispersion;
(3) cell dispersion that makes to step (2) is added the 10ml2%(percent by volume) parting liquid of FBS, behind the mixing at 4 ℃, centrifugal 8 minutes of 600g; Remove supernatant, add 20ml and contain the 20%(percent by volume) the DMEM substratum of BSA, behind the piping and druming mixing, 4 ℃, centrifugal 20 minutes of 1000g; Get undermost precipitation, make the capillary vessel cell;
(4) the capillary vessel cell that step (3) is made is resuspended in the parting liquid of 1ml, the parting liquid that adds again 7.9ml, the DNA enzyme solution of the collagenase of 1ml and 100 μ l, behind the mixing in 37 ℃ of shaking baths (100rpm/min) digestion 90 minutes, make the capillary vessel suspension;
(5) the capillary vessel suspension that step (4) is made is at 4 ℃, centrifugal 5 minutes of 600g; Remove supernatant, precipitation is resuspended in the parting liquid of 2ml, then lightly suspension is placed the top of Percoll concentration gradient centrifugate; 4 ℃, centrifugal 10 minutes of 1000g is divided into six layers of (the first layer: transparent parting liquid; The second layer: transparent Percoll but by some scum silica frost of parting liquid place; The 3rd layer: the cerebrovascular endothelial cell layer that digestion is good; The 4th layer: transparent Percoll; Layer 5: red corpuscle layer; Layer 6: the Percoll crystal), get the 3rd layer of liquid with long lumbar puncture needle, the 3rd layer of liquid is cerebrovascular endothelial cell;
(6) cerebrovascular endothelial cell that step (5) is made is dissolved in the culture fluid of endothelial cell of 1ml, then transfer in the culture plate or 6 orifice plates of 10cm, cultivated 1 day under the normal condition, then use 1 * PBS damping fluid to wash and remove dead cell for twice, with the culture fluid of endothelial cell purifying that contains 3 μ M/ml tetracyclines after 2 days, be replaced by normal cerebrovascular endothelial cell nutrient solution and cultivated 7 days, with 0.01% pancreatin (Typsin) had digestive transfer culture or frozen getting final product;
Described culture plate or 6 orifice plate preparation methods are as follows: use first 1 * Collagen IV bed board 30 minutes, and then with 1 * Fibronectin bed board 30 minutes.
Interpretation of result
1, the present invention in the cerebrovascular endothelial cell nutrient solution except adding endothelial cell growth recruitment factor (ECGS), also add alkaline fiber and given birth to the sub-factor (bFGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-i (IGF-1), endothelium is given birth to the sub-factor (EGF) and vitamins C.These factors can guarantee cellular form, biological nature and physiological function that cerebrovascular endothelium is kept fit and the speed of growth faster; Especially even more important to the growth of senile rat cerebrovascular endothelial cell.If do not add these factors, the senile rat cerebrovascular endothelial cell speed of growth is slow, and cell is relatively more fragile, and is dead easily, can only pass a generation, can not freezing and thawing.
2, culture fluid of endothelial cell of the present invention adopts the serum in PDS(blood plasma source) replaced FBS (foetal calf serum), calf serum (BCS) or horse serum (HS) in traditional nutrient solution, make the cerebrovascular endothelial cell purity of cultivation increase by 30%; Major cause is owing to do not contain the PDGF(platelet-derived growth factor among the FDS), and PDGF is conducive to sneak into the smooth muscle cell in the cerebrovascular endothelial cell, the division of fibrocyte and spongiocyte and propagation; Eliminated this impact with PDS.
3, the present invention has added vitamins C in culture fluid of endothelial cell, heparin and tetracycline, and this makes cerebrovascular endothelial cell grow faster purelyr; Major cause is the growth of vitamins C and heparin stimulating endothelial cell, suppresses the growth of smooth muscle cell; Tetracycline can be suppressed to fibrocellular growth, and these factors have guaranteed the purity of the endotheliocyte of cultivation.
The senile rat cerebrovascular endothelial cell that embodiment 1 described renewal nutrient solution and traditional nutrient solution are cultivated pass 3 generation survival rate the comparison (see figure 1)
Embodiment 2 described senile rat cerebrovascular endothelial cell primary culture methods and traditional method be frozen/recovery after the comparison (see figure 2) of survival rate
Embodiment 2 Comparative Examples
The survival rate 100% in original generation
Frozen/recovery survival rate (78.53 ± 7.76) % (12.45 ± 5.9) %
Embodiment 1 described senile rat cerebrovascular endothelial cell primary culture method and traditional nutrient solution and method cell purity be (see figure 3) relatively
Embodiment 1 Comparative Examples
Cell purity (98.26 ± 1.8) % (68.32 ± 3.2) %
By the above results as can be known, no matter senile rat cerebrovascular endothelial cell primary culture method of the present invention all has significantly raising than prior art at biography 3 generation survival rate, the rear survival rate of frozen/recovery and cell purity.
Claims (2)
1. a senile rat cerebrovascular endothelial cell nutrient solution is characterized in that, on the basis of per hundred milliliters of DMEM substratum, adds following component:
The serum in 20ml-30ml blood plasma source, 100ul penicillin-Streptomycin sulphate solution, 1.0-1.5ml L-glutaminate, 50-150 μ l sulphur fine horse gentamicin, 1.0-1.5ml heparin, 150-300 μ l alkaline fiber is given birth to the sub-factor, 1.0-1.5ml the endothelial cell growth recruitment factor, 200-400 μ l vitamins C, 100-200 μ l vascular endothelial growth factor, 100-200 μ l insulin-like growth factor-i, 100-200 μ l endothelium is given birth to the sub-factor.
2. nutrient solution as claimed in claim 1 is characterized in that, on the basis of per hundred milliliters of DMEM substratum, adds following component:
The serum in 20ml blood plasma source, 100ul penicillin-Streptomycin sulphate solution, 1.0ml L-glutaminate, 100 μ l sulphur fine horse gentamicins, 1.0ml heparin, 150 μ l alkaline fibers are given birth to the sub-factor, 1.0ml the endothelial cell growth recruitment factor, 300 μ l vitamins Cs, 150 μ l vascular endothelial growth factor, 150 μ l insulin-like growth factor-is, 150 μ l endotheliums are given birth to the sub-factor.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673744A (en) * | 2015-03-10 | 2015-06-03 | 王俊力 | In-vitro culturing method for vascular endothelial cells of rat |
| 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 |
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| CN101854941A (en) * | 2007-10-29 | 2010-10-06 | 弗雷森纽斯医疗护理德国有限责任公司 | Use of microvesicles (MVs) derived from stem cells for preparing a medicament for endo/epithelial regeneration of damaged or injured tissues or organs, and related in vitro and in vivo methods |
| CN102641293A (en) * | 2011-02-21 | 2012-08-22 | 杨子江 | Preparation for treating ischemic cerebrovascular diseases and preparation method thereof |
| WO2012121695A1 (en) * | 2011-03-04 | 2012-09-13 | Al-Qahtani Ahmed H | Skin cream |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101854941A (en) * | 2007-10-29 | 2010-10-06 | 弗雷森纽斯医疗护理德国有限责任公司 | Use of microvesicles (MVs) derived from stem cells for preparing a medicament for endo/epithelial regeneration of damaged or injured tissues or organs, and related in vitro and in vivo methods |
| CN102641293A (en) * | 2011-02-21 | 2012-08-22 | 杨子江 | Preparation for treating ischemic cerebrovascular diseases and preparation method thereof |
| WO2012121695A1 (en) * | 2011-03-04 | 2012-09-13 | Al-Qahtani Ahmed H | Skin cream |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673744A (en) * | 2015-03-10 | 2015-06-03 | 王俊力 | In-vitro culturing method for vascular endothelial cells of rat |
| CN104673744B (en) * | 2015-03-10 | 2018-05-25 | 武汉市中西医结合医院 | A kind of vascular endothelial cells extracorporeal culturing method |
| 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 |
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