CN111560345A - Method for separating and purifying primary valvular endothelial cells of pigs - Google Patents
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Abstract
The invention discloses a method for separating and purifying primary valve endothelial cells of a pig, which comprises the following steps: step 1, early preparation, namely preparing medical instruments and a cell culture standard; step 2, coating, namely adding the prepared coating solution into a culture dish respectively, incubating for at least 1 hour at 37 ℃, and washing with PBS for 3 times before cell seeding; step 3, taking the valve tissue to shear the pig valve tissue in a sterile environment, and putting the pig valve tissue into pre-cooled sterile heparin normal saline at the temperature of 4 ℃ to wash the pig valve tissue; and 4, layering and separating the cells through the configured density gradient liquid under the action of a gravity or centrifugal force field. The invention has the following beneficial effects: the method for separating and purifying the primary porcine valve endothelial cells lasts for about 2 hours, the separating and purifying efficiency of the valve endothelial cells is greatly improved, and the endothelial cells can be observed to be arranged and grow in a vortex shape or a paving stone shape after 3-4 hours; the purity of endothelial cells is more than 95% as shown by immunofluorescence, and the method is simple and easy to operate.
Description
Technical Field
The invention relates to the field of bioengineering, and in particular relates to a method for separating and purifying porcine primary valve endothelial cells.
Background
Porcine heart valve endothelial cells were isolated from heart valve tissue with valves between the atria and ventricles, and between the ventricles and the vessels leaving the ventricles. The left atrium and the right atrium and the left ventricle are separated by intervals and are not communicated with each other, and valves (atrioventricular valves) are arranged between the atria and the ventricles and are equivalent to guards, so that blood can only flow into the ventricles from the atria but can not flow backwards.
The valve endothelial cells are important tool cells in the field of tissue engineering valves, and how to obtain high-purity valve endothelial cells is always the key and difficult point of in vitro research of tissue engineering. At present, for the isolated culture of primary endothelial cells, an enzyme digestion method, a physical scraping method, a screening method combined with an immunomagnetic bead sorting method or a medicine purification method is generally adopted at home and abroad, but the problems of low purity, low yield, complex operation steps, time and labor waste and the like exist all the time. Although the endothelial cell lines of different germ line sources are easy to obtain, the endothelial cell lines are different from primary endothelial cells in certain biological characters and phenotypes due to genetic modification, and cannot completely simulate the morphological structure and the function of the tissue endothelial cells.
Disclosure of Invention
Technical problem to be solved
The invention aims to provide a method for separating and purifying primary valvular endothelial cells of a pig, which aims to solve the problems in the background technology.
(II) technical scheme
In order to realize the purpose of solving the problems of the background technology by the separation and purification method of the primary valvular endothelial cells of the pigs, the invention provides the following technical scheme:
a method for separating and purifying primary valvular endothelial cells of pigs comprises the following steps:
step 1, early preparation
1. Sterilizing medical instruments under high pressure, placing in a centrifuge tube filled with 75% alcohol for 20min, and naturally air drying in a culture dish;
2. putting gauze on the workbench;
3. placing the endothelial cell culture medium in a water bath at 37 ℃;
step 2, coating
The common coating liquid comprises Laminin (Laminin), polylysine (Poly-D-Lysine) and Gelatin, wherein one of the three can be selected as the coating liquid,
laminin was diluted with 1 Xbuffer, 10ug/ml as the working concentration;
preparing polylysine into 1.5% solution with sterile double distilled water;
the gelatin is prepared into 1.5% solution with sterile double distilled water, and is used after micro-boiling by microwave heating.
Respectively adding the prepared coating solution into a culture dish, incubating for at least 1 hour at 37 ℃, and washing with PBS 3 times before cell seeding; the coating proportion is as follows: 0.5ml/12 well plate, 1ml/6 well plate, 1 ml/3 cm counting well and 3ml/10cm dish;
step 3, taking valve tissue
Shearing the porcine valve tissue in a sterile environment, and putting the porcine valve tissue into pre-cooled sterile heparin normal saline at 4 ℃ for washing;
step 4, digestion
1. Adding 10ml of type I collagenase solution with the concentration of 0.1% into a 15ml centrifuge tube, and shaking for 60min at the temperature of 37 ℃;
2. taking the supernatant, and putting the supernatant into 2ml of serum to be mixed uniformly;
3. mixing the supernatant, filtering, centrifuging at 800rpm and 4 deg.C for 5min, re-suspending with 5ml buffer solution, and adding the cell suspension into 15ml centrifuge tube;
4. slowly adding the resuspended cells into the prepared density gradient liquid, centrifuging for 35min at the rotating speed of 3000rpm and the temperature of 4 ℃, wherein the liquid surface is divided into two layers after centrifugation, the upper layer is an endothelial cell layer, the middle layer is a mesenchymal cell layer, and the bottom layer of a centrifuge tube is a red blood cell layer;
5. sucking the epithelial cell layer in the upper layer, filtering by a cell sieve, centrifuging for 5min at the rotating speed of 800rpm and the temperature of 4 ℃, resuspending, counting and plating.
Preferably, the medical instrument of step 1 comprises 2 pairs of ophthalmic scissors and 2 pairs of fine ophthalmic tweezers.
Preferably, the coating solution of step 2 is Laminin (Laminin), polylysine (Poly-D-Lysine) or Gelatin (Gelatin), and any one of the three may be used as the coating solution.
Preferably, the endothelial cell culture medium of step 1 is 50mL of endothelial cell culture medium, which comprises: 44mL of F12 medium, 5mL of FBS, 500. mu.L of 100 Xcyan-streptomycin, 1.5mg of endothelial cell growth supplement, 250U of heparin, 125. mu.g of ascorbic acid, 500. mu.L of glutamine, stored at 4 ℃.
Preferably, the buffer solution of step 4 is prepared from the following components:
| serial number | Composition (I) | MW | 1Liter (1X buffer) | 1Liter (5X buffer) |
| 1 | Nacl | 58.44 | 8g | 40g |
| 2 | Kcl | 74.55 | 0.4g | 2g |
| 3 | MgSO4.7H2O | 246 | 0.2g | 1g |
| 4 | Glucose | 180.2 | 1g | 5g |
| 5 | KH2PO4 | 136.9 | 60mg | 300mg |
| 6 | Na2HPO4 | 142.0 | 48.3mg | 241.5mg |
| 7 | HEPES | 238.3 | 4.766g | 23.83g |
Preparing the above 1X buffer solution and 5X buffer solution, diluting with ultrapure water to constant volume, adjusting pH to 7.5 with NaOH, filtering with 0.22um filter, and storing at 4 deg.C.
Preferably, the density gradient liquid of step 4 comprises the following components: solution A, 10ml of 45% high-density solution; solution B, 10ml of 22% low-density solution; wherein the component of the solution A is as follows: 3.5ml of D2H2O, 4.5ml of percoll, 2ml of 5 Xbuffer; the liquid B comprises the following components: 5.8ml of D2H2O, 2.2ml of percoll, 2ml of 5 Xbuffer; dripping the solution A into a 15ml centrifuge tube, slowly adding the solution B without shaking the liquid surface, and preparing the density gradient solution with the volume ratio of the solution A to the solution B being 4: 4.
(III) advantageous effects
Compared with the prior art, the invention provides a method for separating and purifying primary porcine valve endothelial cells, which has the following beneficial effects: the method for separating and purifying the primary porcine valve endothelial cells lasts for about 2 hours, the separating and purifying efficiency of the valve endothelial cells is greatly improved, and the endothelial cells can be observed to be arranged and grow in a vortex shape or a paving stone shape after 3-4 hours; the purity of endothelial cells is more than 95% as shown by immunofluorescence, and the method is simple and easy to operate. The concrete expression is as follows:
the method has the advantages that the separation effect is good, the separation time is short, endothelial cells with higher purity and higher quantity can be obtained at one time, and the growth of interstitial cells is not needed to be interfered by medicines in the later period;
wide application range, can separate cells with sedimentation coefficient difference like a centrifugal method at high speed, and can separate cells with certain buoyancy density difference;
③ the cells will not be extruded and deformed, the activity of the cells can be maintained, and the formed zones can be prevented from mixing due to convection.
Drawings
FIG. 1 is a schematic overall flow diagram of the present invention;
FIG. 2 is a schematic diagram of a preliminary preparation process of the present invention;
FIG. 3 is a schematic diagram of a digestion process according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a method for separating and purifying primary valve endothelial cells of a pig, which comprises the following steps:
step 1, early preparation
1. Sterilizing medical instruments under high pressure, placing in a centrifuge tube filled with 75% alcohol for 20min, and naturally air drying in a culture dish;
2. putting gauze on the workbench;
3. placing the endothelial cell culture medium in a water bath at 37 ℃;
step 2, coating
Respectively adding the prepared coating solution into a culture dish, incubating for at least 1 hour at 37 ℃, and washing with PBS 3 times before cell seeding; the coating proportion is as follows: 0.5ml/12 well plate, 1ml/6 well plate, 1 ml/3 cm counting well and 3ml/10cm dish;
step 3, taking valve tissue
Shearing the porcine valve tissue in a sterile environment, and putting the porcine valve tissue into pre-cooled sterile heparin normal saline at 4 ℃ for washing;
step 4, digestion
1. Adding 10ml of type I collagenase solution with the concentration of 0.1% into a 15ml centrifuge tube, and shaking for 60min at the temperature of 37 ℃;
2. taking the supernatant, and putting the supernatant into 2ml of serum to be mixed uniformly;
3. mixing the supernatant, filtering, centrifuging at 800rpm and 4 deg.C for 5min, re-suspending with 5ml buffer solution, and adding the cell suspension into 15ml centrifuge tube;
4. slowly adding the resuspended cells into the prepared density gradient liquid, centrifuging for 35min at the rotating speed of 3000rpm and the temperature of 4 ℃, wherein the liquid surface is divided into two layers after centrifugation, the upper layer is an endothelial cell layer, the middle layer is a mesenchymal cell layer, and the bottom layer of a centrifuge tube is a red blood cell layer;
5. sucking the epithelial cell layer in the upper layer, filtering by a cell sieve, centrifuging for 5min at the rotating speed of 800rpm and the temperature of 4 ℃, resuspending, counting and plating.
Wherein, the medical apparatus in the step 1 comprises 2 pairs of ophthalmic scissors and 2 pairs of fine ophthalmic tweezers.
The coating liquid in the step 2 is Laminin (Lamin), polylysine (Poly-D-Lysine) or Gelatin (Gelatin), and any one of the three can be used as the coating liquid.
The endothelial cell culture medium of step 1 is 50mL of endothelial cell culture medium, comprising: 44mL of F12 medium, 5mL of FBS, 500. mu.L of 100 Xcyan-streptomycin, 1.5mg of endothelial cell growth supplement, 250U of heparin, 125. mu.g of ascorbic acid, 500. mu.L of glutamine, stored at 4 ℃.
The buffer solution preparation component of the step 4 is:
| serial number | Composition (I) | MW | 1Liter (1X buffer) | 1Liter (5X buffer) |
| 1 | Nacl | 58.44 | 8g | 40g |
| 2 | Kcl | 74.55 | 0.4g | 2g |
| 3 | MgSO4.7H2O | 246 | 0.2g | 1g |
| 4 | Glucose | 180.2 | 1g | 5g |
| 5 | KH2PO4 | 136.9 | 60mg | 300mg |
| 6 | Na2HPO4 | 142.0 | 48.3mg | 241.5mg |
| 7 | HEPES | 238.3 | 4.766g | 23.83g |
Preparing the above 1X buffer solution and 5X buffer solution, diluting with ultrapure water to constant volume, adjusting pH to 7.5 with NaOH, filtering with 0.22um filter, and storing at 4 deg.C.
The density gradient liquid in the step 4 comprises the following components: solution A, 10ml of 45% high-density solution; solution B, 10ml of 22% low-density solution; wherein the component of the solution A is as follows: 3.5ml of D2H2O, 4.5ml of percoll, 2ml of 5 Xbuffer; the liquid B comprises the following components: 5.8ml of D2H2O, 2.2ml of percoll, 2ml of 5 Xbuffer; dripping the solution A into a 15ml centrifuge tube, slowly adding the solution B without shaking the liquid surface, and preparing the density gradient solution with the volume ratio of the solution A to the solution B being 4: 4.
In summary, the invention uses a certain medium to form a discontinuous density gradient in a centrifuge tube, the cell suspension is placed on the top of the medium, the cells are layered and separated under the action of gravity or a centrifugal force field, the cells of the valve are mainly divided into endothelial cells and interstitial cells, the endothelial cells and the interstitial cells have certain difference in terms of cell density, cell granularity and cell buoyancy, and the two cells can be well divided into two gradients through the selection of the density gradient and the density medium. The density gradient medium has the following advantages: firstly, density gradient can be generated, and when the density is high, the viscosity is not high; the PH is easy to adjust to be neutral; repeated attempts of different density gradients show that the osmotic pressure does not change greatly when the density medium is high, the density medium is basically isotonic and does not cause cell deformation, and the separation technology comprises the following steps: the separation effect is good, the separation time is short (30min), endothelial cells with higher purity (95%) and higher quantity can be obtained at one time, and no medicament is needed to interfere the growth of interstitial cells in the later period; wide application range, can separate cells with sedimentation coefficient difference like a fast centrifugation method, and can separate cells with certain buoyancy density difference; ③ the cells will not be extruded and deformed, the activity of the cells can be maintained, and the formed zones can be prevented from mixing due to convection.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. A method for separating and purifying primary valvular endothelial cells of pigs is characterized by comprising the following steps:
step 1, early preparation
1. Sterilizing medical instruments under high pressure, placing in a centrifuge tube filled with 75% alcohol for 20min, and naturally air drying in a culture dish;
2. putting gauze on the workbench;
3. placing the endothelial cell culture medium in a water bath at 37 ℃;
step 2, coating
Respectively adding the prepared coating solution into a culture dish, incubating for at least 1 hour at 37 ℃, and washing with PBS 3 times before cell seeding; the coating proportion is as follows: 0.5ml/12 well plate, 1ml/6 well plate, 1 ml/3 cm counting well and 3ml/10cm dish;
step 3, taking valve tissue
Shearing the porcine valve tissue in a sterile environment, and putting the porcine valve tissue into pre-cooled sterile heparin normal saline at 4 ℃ for washing;
step 4, digestion
1. Adding 10ml of type I collagenase solution with the concentration of 0.1% into a 15ml centrifuge tube, and shaking for 60min at the temperature of 37 ℃;
2. taking the supernatant, and putting the supernatant into 2ml of serum to be mixed uniformly;
3. mixing the supernatant, filtering, centrifuging at 800rpm and 4 deg.C for 5min, re-suspending with 5ml buffer solution, and adding the cell suspension into 15ml centrifuge tube;
4. slowly adding the resuspended cells into the prepared density gradient liquid, centrifuging for 35min at the rotating speed of 3000rpm and the temperature of 4 ℃, wherein the liquid surface is divided into two layers after centrifugation, the upper layer is an endothelial cell layer, the middle layer is a mesenchymal cell layer, and the bottom layer of a centrifuge tube is a red blood cell layer;
5. sucking the epithelial cell layer in the upper layer, filtering by a cell sieve, centrifuging for 5min at the rotating speed of 800rpm and the temperature of 4 ℃, resuspending, counting and plating.
2. The method for separating and purifying porcine primary valve endothelial cells according to claim 1, wherein the method comprises the following steps: the medical apparatus of the step 1 comprises 2 pairs of ophthalmic scissors and 2 pairs of fine ophthalmic tweezers.
3. The method for separating and purifying porcine primary valve endothelial cells according to claim 1, wherein the method comprises the following steps: the coating solution in the step 2 is Laminin (Laminin), polylysine (Poly-D-Lysine) or Gelatin (Gelatin), and one of the Laminin, the polylysine and the Gelatin is selected as the coating solution.
4. The method for separating and purifying porcine primary valve endothelial cells according to claim 1, wherein the method comprises the following steps: the endothelial cell culture medium of the step 1 is 50mL of endothelial cell culture medium, and comprises: 44mL of F12 medium, FBS5mL, 500. mu.L of 100 Xcyan-streptomycin, 1.5mg of endothelial cell growth supplement, 250U of heparin, 125. mu.g of ascorbic acid, 500. mu.L of glutamine, stored at 4 ℃.
5. The method for separating and purifying porcine primary valve endothelial cells according to claim 1, wherein the method comprises the following steps: the buffer solution preparation components of the step 4 are as follows:
Preparing the above 1X buffer solution and 5X buffer solution, diluting with ultrapure water to constant volume, adjusting pH to 7.5 with NaOH, filtering with 0.22um filter, and storing at 4 deg.C.
6. The method for separating and purifying porcine primary valve endothelial cells according to claim 1, wherein the density gradient solution of step 4 comprises the following components: solution A, 10ml of 45% high-density solution; solution B, 10ml of 22% low-density solution; wherein the component of the solution A is as follows: 3.5ml of D2H2O, 4.5ml of percoll, 2ml of 5 Xbuffer; the liquid B comprises the following components: 5.8ml of D2H2O, 2.2ml of percoll, 2ml of 5 Xbuffer; dripping the solution A into a 15ml centrifuge tube, slowly adding the solution B without shaking the liquid surface, and preparing the density gradient solution with the volume ratio of the solution A to the solution B being 4: 4.
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