CN103045534B - Aged rat brain vascular endothelial cell culture fluid - Google Patents

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

A kind of old rat cerebral vascular endothelial cell culture medium

technical field

The invention relates to a culture medium of cerebrovascular endothelial cells of aged rats, which belongs to the field of biotechnology.

Background technique

The existing primary culture of rat cerebrovascular endothelial cells is obtained from neonatal rats (<10 days, 35-50g), juvenile rats (<3weeks, 80-100g) or young adult rats (2-3 months old, 200g- 300g), and the cultured cerebrovascular endothelial cells can only be passaged once and cannot be frozen. Using rat cerebrovascular endothelial cells as experimental materials leads to the disadvantages of long time and high experimental cost. Such 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.

The precedent of successful culture of cerebrovascular endothelial cells in aged rats (19-22 months, 900g-1200g) has not been reported so far; the existing material objects and culture techniques cannot meet the requirements of establishing the aged blood-brain barrier model in vitro and studying the effects of the aged blood-brain barrier. Physiological properties, and requirements for blood-brain barrier-related degeneration in geriatric neurological disease.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a culture medium for cerebrovascular endothelial cells of aged rats.

Technical scheme of the present invention is as follows:

A kind of old rat cerebral vascular endothelial cell culture fluid, on the basis of every 100 milliliters of DMEM culture medium, add the following components:

20ml-30ml plasma-derived serum, 100ul penicillin-streptomycin solution, 1.0-1.5ml L-glutamine, 50-150μl sulfur gentamicin, 1.0-1.5ml heparin, 150-300μl basic fibrogenic factor , 1.0-1.5ml endothelial cell growth supplement factor, 200-400μl vitamin C, 100-200μl vascular endothelial growth factor, 100-200μl insulin-like growth factor-1, 100-200μl endothelial factor.

Add the above components to the DMEM medium.

Preferably according to the present invention, on the basis of per 100 milliliters of DMEM medium, add the following components:

20ml of plasma-derived serum, 100ul of penicillin-streptomycin solution, 1.0ml of L-glutamine, 100μl of sulfur gentamicin, 1.0ml of heparin, 150μl of basic fibroblast factor, 1.0ml of endothelial cell growth supplementation factor (ECGS ), 300 μl vitamin C, 150 μl vascular endothelial growth factor (VEGF), 150 μl insulin-like growth factor-1 (IGF-1), 150 μl endothelial growth factor (EGF).

The above reagents are commercially available products commonly used in this field.

Beneficial effect

1. The endothelial cell culture medium of the present invention adopts PDS (plasma-derived serum) to replace FBS (fetal bovine serum), calf serum (BCS) or horse serum (HS) in traditional culture medium, so that the cultured cerebrovascular The purity of endothelial cells increased by 30%; the main reason is that PDGF (platelet-derived growth factor) is not contained in FDS, and PDGF is beneficial to the division and proliferation of smooth muscle cells, fibroblasts and glial cells mixed in cerebrovascular endothelial cells; PDS eliminates this effect.

2. The present invention adds vitamin C and puromycin to the endothelial cell culture medium, which makes the cerebrovascular endothelial cells grow faster and more purely.

3. The present invention also adds vascular endothelial growth factor, insulin-like growth factor-1, and endothelial factor to the endothelial cell culture medium. These factors can make the cerebrovascular endothelial cells grow faster and healthier, especially for aged rats The growth of cerebrovascular endothelial cells is more important.

Description of drawings

Fig. 1, the aged rat cerebrovascular endothelial cell that the present invention makes through primary culture pass 3 generations and the old rat cerebrovascular endothelial cell primary culture that traditional method prepares the columnar comparative figure of the survival rate of pass 3 generations;

Fig. 2, the columnar comparison chart of the survival rate of aged rat cerebrovascular endothelial cells prepared by the traditional method after cryopreservation and resuscitation of aged rat cerebrovascular endothelial cells prepared by the present invention;

Fig. 3: A columnar graph comparing the purity of aged rat cerebrovascular endothelial cells prepared by the present invention and aged rat cerebrovascular endothelial cells prepared by traditional methods.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto.

Raw material source:

19-22-month-old rats were purchased from Beijing Weitong Lihua Experimental Animal Technology Center;

Sources of various reagents for preparing aged rat cerebrovascular endothelial cell culture medium:

DMEM medium, purchased from Gibico Company, product number 11995-065;

Plasma-derived serum (PDS, Plasma-Derived Serum), purchased from Bioquote, product number BT-214-10;

Penicillin-streptomycin solution, purchased from sigma company, product number p0718;

L-Glutamine, available from Sigma, product number G7513;

Sulfur gentamicin, purchased from sigma company, product number G1397;

Heparin, purchased from Sigma, product number H3149;

Basic fibrogenesis factor (bFGF), purchased from Sigma Company, product number F9786;

Endothelial cell growth supplement factor (ECGS), purchased from Sigma, product number E2759;

Vitamin C, purchased from Sigma Company, product number c1768;

Vascular endothelial growth factor (VEGF), purchased from Lonza Company, product number cc4176;

Insulin-like growth factor-1 (IGF-1) was purchased from Lonza Company, product number cc4176;

Endothelial factor (EGF) was purchased from Lonza Company, product number cc4176;

Puromycin (Puromycin) was purchased from Sigma Company, product number p8833.

Sources of various enzymes and other related reagents:

Fetal bovine serum (FBS) was purchased from Invitrogen Company, product number 10437-028;

Collagenase (Collagenase) was purchased from Invitrogen, product number 17101;

DNase (DNase) was purchased from Sigma-Aldrich Company, product number D4263;

Trypsin (Typsin) was purchased from Sigma-Aldrich Company, product number T3053;

Bovine serum albumin (BSA) was purchased from Sigma-Aldrich, product number a3059;

Collagen IV (Collagen IV) was purchased from Sigma-Aldrich Company, product number C5533:

Fibronectin (Fibronectin) was purchased from Sigma-Aldrich Company, product number F1141;

Percoll was purchased from Sigma-Aldrich Company, product number 4937;

Lumbar puncture needle (Lumbar puncture needle) was purchased from BD Company, product number 405182.

Example 1

A kind of old rat cerebral vascular endothelial cell culture fluid, on the basis of every 100 milliliters of DMEM culture medium, add the following components:

30ml of plasma-derived serum, 100μl of penicillin-streptomycin solution, 1.0ml of L-glutamine, 100μl of gentamycin, 1.5ml of heparin, 150μl of basic fibrogenic factor, 1.0ml of endothelial cell growth supplementation factor, 400μl Vitamin C, 200 μl vascular endothelial growth factor, 200 μl insulin-like growth factor-1, 200 μl endothelial factor;

Add the above components to the DMEM medium.

Example 2

A kind of old rat cerebral vascular endothelial cell culture fluid, on the basis of every 100 milliliters of DMEM culture medium, add the following components:

20ml of plasma-derived serum, 100μl of penicillin-streptomycin solution, 1.0ml of L-glutamine, 100μl of gentamycin, 1.0ml of heparin, 100μl of basic fibrogenic factor, 1.0ml of endothelial cell growth supplementation factor, 200μl Vitamin C, 100 μl vascular endothelial growth factor, 100 μl insulin-like growth factor-1, 100 μl endothelial factor;

Add the above components to the DMEM medium.

comparative example

The traditional cerebrovascular endothelial cell culture medium is to add the following components on the basis of every 100ml of F12 medium (Ham’s-F12 medium):

10-20ml calf serum (BCS), 100μl penicillin-streptomycin solution. (This formulation is recorded in Bowman PD, et al. Primary Culture of Capillary Endothelium From Rat Brain. INVITRO.1981.17(4), 353-362).

or it could be:

44ml DMEM medium, 44ml Ham’s-F12 medium, 10ml fetal bovine serum (FBS), 100μl penicillin-streptomycin, 1.0ml heparin sodium, 1.0ml endothelial cell growth supplement factor. (The formula is recorded in Tunkel AR et al. Blood-brain barrier alterations in bacterial meningitis: development of an in vitro model and observations on the effects of lipopolysaccharide. In Vitro Cell Dev Biol. 199127A(2): 113-20).

Test case

The above-mentioned nutrient solution was experimentally cultured as follows:

(1) Decapitate 20-month-old aged rats, peel off the skin of the rat's head, and then soak the rat's head in a separation solution containing 2% (volume percent) fetal bovine serum (FBS) at 0°C for 5 Minutes, take it out, cut the two cerebral hemispheres, roll the two cerebral hemispheres on sterilized ordinary filter paper for a week, then remove the brainstem and white matter, and keep the gray matter to obtain the sample tissue;

The separation solution is based on the DMEM medium, and the following components are added per 100 ml: 1 ml penicillin-streptomycin solution; 100 μl thiopurine gentamycin;

(2) Place the sample tissue in a 5ml ice-cold petri dish containing 2% (volume percent) FBS separation solution and cut it into small pieces of sample tissue with a size of ^1-2mm3 , add 2% (volume percent) FBS separation solution to 25ml , dispersed by pipetting, then add 2.5ml of collagenase and 250μl of DNase solution to the sample tissue fragments dispersed by pipetting, and digest in a water bath shaker at 36°C at 100rpm/min for 90 minutes; then continue to digest for 30 minutes after being dispersed by pipetting Minutes to make dispersed cells;

(3) Add 10ml of 2% (volume percentage) FBS separation solution to the dispersed cells prepared in step (2), mix well and centrifuge at 4°C, 600g for 8 minutes; remove the supernatant, add 20ml containing 20% (volume percentage) Percentage) DMEM medium of BSA, after mixing by pipetting, centrifuge at 1000g for 20 minutes at 4°C; take the bottom layer of precipitate to obtain capillary cells;

(4) Resuspend the capillary cells prepared in step (3) in 1ml of separation solution, then add 7.9ml of separation solution, 1ml of collagenase and 100μl of DNase solution, mix well and shake in a water bath at 37°C Digest in bed (100rpm/min) for 90 minutes to obtain capillary suspension;

(5) Centrifuge the capillary suspension prepared in step (4) at 4°C, 600g for 5 minutes; remove the supernatant, resuspend the pellet in 2ml of separation medium, and then gently place the suspension in Percoll concentration The top of the gradient centrifugation solution; 4°C, 1000g centrifugation for 10 minutes, divided into six layers (first layer: transparent separation liquid; second layer: transparent Percoll but some scum near the separation liquid; third layer: digestion Good cerebral vascular endothelial cell layer; fourth layer: transparent Percoll; fifth layer: red blood cell layer; sixth layer: Percoll crystal), use a long lumbar puncture needle to take the third layer of liquid, the third layer of liquid is the brain Vascular endothelial cells;

(6) Dissolve the cerebrovascular endothelial cells prepared in step (5) in 1ml of endothelial cell culture medium, then transfer to a 10cm culture plate or a 6-well plate, culture for 1 day under normal conditions, and then wash with 1×PBS Wash twice with buffer to remove dead cells, purify with endothelial cell culture medium containing 3 μM/ml puromycin for 2 days, replace with normal cerebrovascular endothelial cell culture medium and culture for 7 days, digest with 0.01% trypsin (Typsin) Passage or cryopreservation;

The preparation method of the culture plate or 6-well plate is as follows: 1×Collagen IV was first used to spread the plate for 30 minutes, and then 1×Fibronectin was used to spread the plate for 30 minutes.

Result analysis

1. The present invention adds basic fibroblast factor (bFGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor-1 to the cerebrovascular endothelial cell culture fluid in addition to adding endothelial cell growth supplement factor (ECGS). (IGF-1), endothelial factor (EGF) and vitamin C. These factors can ensure that the cerebrovascular endothelium maintains healthy cell morphology, biological characteristics, physiological functions and rapid growth rate; it is especially important for the growth of cerebrovascular endothelial cells in aged rats. If these factors are not added, the growth rate of the cerebrovascular endothelial cells of aged rats is slow, the cells are relatively fragile and easy to die, and can only be passed on to one generation, and cannot be frozen and revived.

2. The endothelial cell culture medium of the present invention adopts PDS (plasma-derived serum) to replace FBS (fetal bovine serum), calf serum (BCS) or horse serum (HS) in traditional culture medium, so that the cultured cerebrovascular The purity of endothelial cells increased by 30%; the main reason is that PDGF (platelet-derived growth factor) is not contained in FDS, and PDGF is beneficial to the division and proliferation of smooth muscle cells, fibroblasts and glial cells mixed in cerebrovascular endothelial cells; PDS eliminates this effect.

3. The present invention adds vitamin C, heparin and puromycin to the endothelial cell culture medium, which makes the cerebrovascular endothelial cells grow faster and more purely; the main reason is that vitamin C and heparin stimulate the growth of endothelial cells and inhibit smooth muscle cells growth; puromycin can inhibit the growth of fibroblasts, and these factors ensure the purity of cultured endothelial cells.

Comparison of the survival rate of aged rat cerebrovascular endothelial cells cultured in the updated culture medium described in Example 1 and the traditional culture medium for three passages (see Figure 1)

Comparison of the survival rate between the primary culture method of aged rat cerebrovascular endothelial cells described in Example 2 and the traditional method after cryopreservation/resuscitation (see Figure 2)

Example 2 Comparative example

The survival rate of the original generation is 100%

Cryopreservation/resuscitation survival rate (78.53±7.76)% (12.45±5.9)%

Comparison of cell purity between the primary culture method of aged rat cerebrovascular endothelial cells described in Example 1 and the traditional culture medium and method (see Figure 3)

Example 1 Comparative example

Cell Purity (98.26±1.8)% (68.32±3.2)%

From the above results, it can be seen that the method for primary culture of aged rat cerebrovascular endothelial cells of the present invention has greatly improved the survival rate of 3 passages, the survival rate after cryopreservation/resuscitation and cell purity compared with the prior art.

Claims (2)

1. an aged rat cerebrovascular endothelial cell culture fluid is characterized in that, on the basis of every 100 milliliters of DMEM medium, add following components: 20ml-30ml plasma-derived serum, 100ul penicillin-streptomycin solution, 1.0-1.5ml L-glutamine, 50-150μl sulfur gentamicin, 1.0-1.5ml heparin, 150-300μl basic fibrogenic factor , 1.0-1.5ml endothelial cell growth supplement factor, 200-400μl vitamin C, 100-200μl vascular endothelial growth factor, 100-200μl insulin-like growth factor-1, 100-200μl endothelial factor. 2. culture fluid as claimed in claim 1, is characterized in that, on the basis of every 100 milliliters of DMEM medium, add following component: 20ml of plasma-derived serum, 100ul of penicillin-streptomycin solution, 1.0ml of L-glutamine, 100μl of gentamycin, 1.0ml of heparin, 150μl of basic fibroblast factor, 1.0ml of endothelial cell growth supplementation factor, 300μl Vitamin C, 150 μl vascular endothelial growth factor, 150 μl insulin-like growth factor-1, 150 μl endothelial factor.