CN110295144B - Method for separating and extracting primary neurons of bladder - Google Patents
Method for separating and extracting primary neurons of bladder Download PDFInfo
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- CN110295144B CN110295144B CN201910639568.0A CN201910639568A CN110295144B CN 110295144 B CN110295144 B CN 110295144B CN 201910639568 A CN201910639568 A CN 201910639568A CN 110295144 B CN110295144 B CN 110295144B
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Abstract
The invention provides a method for separating and extracting primary neurons of a bladder, which relates to the technical field of bioengineering. According to the method provided by the invention, the primary bladder neurons are successfully treated from the bladder body by optimizing the aspects of material taking, digestion and culture, microscopic examination identification can be realized by using various methods, the cell activity of the primary bladder neurons is well ensured, and the test requirement that rat bladder primary neuron cells are used as test objects is met. Successful separation and extraction of primary neurons of the bladder can lay a foundation for further research on neurogenic lesions of the bladder.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for separating and extracting primary neurons of bladder.
Background
Primary cell culture, also called primary culture, is the first step in establishing cell lines and includes the steps of material selection, separation, culture, maintenance, etc. The culture of the primary cells can provide powerful means for the growth, metabolism and propagation of biological cells, can create conditions for subculture, and is also an essential step for clinical practice and drug screening.
At present, there are many kinds of primary cell cultures of bladder, such as primary cells of bladder cancer, primary fibroblasts of bladder, primary smooth muscle cells of bladder, but there is no research on the extraction and culture of primary neurons of bladder. The reason is that the number of neurons in bladder tissues is small, and the bladder neurons are combined with smooth muscle cells and epithelial cells, so that the bladder neuron cells are difficult to separate, and much trouble is brought to research work. Moreover, there is usually damage to neurons during the process of bladder material selection, digestion and culture, so that other non-neuronal cells dominate the growth during culture to inhibit the growth of neurons, and so on, so that an effective method for separating and extracting primary neurons in bladder is lacking at present.
Disclosure of Invention
The invention provides a method for separating and extracting primary neurons of a bladder, aiming at overcoming the defect that the prior art lacks a method for separating and extracting the primary neurons of the bladder.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for separating and extracting primary neurons of bladder, which comprises the following steps:
(1) taking a bladder body, washing the bladder body by sterile Kirschner liquid, and shearing the bladder body to obtain a pretreated bladder sample;
(2) pretreating the bladder sample by digestion with type II collagenase, centrifuging and discarding the supernatant to obtain a first digest;
(3) digesting the first digestion product with trypsin for 3-7 min, centrifuging and removing a supernatant to obtain a second digestion product;
(4) mixing the second digest with a neuron complete culture medium, filtering by a cell filter, carrying out shake culture on a filtrate for 20-40 min, centrifuging, removing a supernatant, carrying out heavy suspension on the obtained precipitate by using the neuron complete culture medium, adding the heavy suspension to a cell slide coated with poly-D-lysine and laminin in advance, culturing for 4-7D, carrying out full liquid change after 48h of culture, and carrying out full liquid change again after 48-96 h of culture;
the neuron complete culture medium takes a Neurobasal-A culture medium as a basal culture medium, and further comprises: 1-3% of B-27 type serum-free additive, 0.2-2% of L-glutamine, 0.05-0.2% of GDNF and 1-5% of 100 multiplied antibacterial-antifungal agent.
Preferably, in the step (2), the concentration of the type II collagenase is 1 to 5mg/ml, and the ratio of the volume of the type II collagenase to the mass of the pretreated bladder sample is 1 to 5 ml: 1g of the total weight of the composition.
Preferably, in the step (2), the digestion time of the type II collagenase is 1-2 min.
Preferably, in the step (3), when the trypsin digestion is terminated, the digestion is terminated by adding a buffer culture medium at 4 ℃;
the buffer culture medium takes an F12 culture medium as a basic culture medium, and further comprises: fetal calf serum with the mass concentration of 5-12% and 100 multiplied by antibacterial-antifungal agent with the mass concentration of 0.5-3%.
Preferably, in step (3), the amount of the buffer medium added is more than 2 times the total volume of the trypsin and the first digest.
Preferably, the inner diameter of the cell filter is 40-100 μm.
Preferably, in steps (2) and (3), the digestion is carried out at 37 ℃ and 5% CO2And (5) digesting in a shaking table in an incubator.
Preferably, in the step (3), the ratio of the mass of the pretreated bladder sample to the volume of the neuron complete medium is 1 g: mixing the second digest with a neuron complete medium at a ratio of 0.1-2 ml.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for separating and extracting primary neurons of bladder, which comprises the steps of washing and shearing bladder bodies, sequentially digesting the bladder bodies by type II collagenase and trypsin, filtering obtained digest cells, inoculating a filtrate into a complete neuron culture medium for culture, and separating to obtain the primary neurons of bladder. According to the method provided by the invention, the primary bladder neurons are successfully treated from the bladder body by optimizing the aspects of material taking, digestion and culture, microscopic examination identification can be realized by using various methods, the cell activity of the primary bladder neurons is well ensured, and the test requirement that rat bladder primary neuron cells are used as test objects is met. Successful separation and extraction of primary neurons of the bladder can lay a foundation for further research on neurogenic lesions of the bladder.
Drawings
FIG. 1 is a photograph of primary neuronal cells of rat bladder observed by an optical microscope in example 2;
FIG. 2 is another photograph of primary neuronal cells of rat bladder observed by light microscope in example 2;
FIG. 3 is a photograph of primary neurons of rat bladder observed by confocal laser microscopy in example 2;
FIG. 4 is another photograph of primary neurons from rat bladder observed by confocal laser microscopy as in example 2.
Detailed Description
The invention provides a method for separating and extracting primary neurons of bladder, which comprises the following steps:
(1) taking a bladder body, washing the bladder body by sterile Kirschner liquid, and shearing the bladder body to obtain a pretreated bladder sample;
(2) pretreating the bladder sample by digestion with type II collagenase, centrifuging and discarding the supernatant to obtain a first digest;
(3) digesting the first digestion product with trypsin for 3-7 min, centrifuging and removing a supernatant to obtain a second digestion product;
(4) mixing the second digest with a neuron complete culture medium, filtering by a cell filter, carrying out shake culture on a filtrate for 20-40 min, centrifuging, removing a supernatant, carrying out heavy suspension on the obtained precipitate by using the neuron complete culture medium, adding the heavy suspension to a cell slide coated with poly-D-lysine and laminin in advance, culturing for 4-7D, carrying out full liquid change after 48h of culture, and carrying out full liquid change again after 48-96 h of culture;
the neuron complete culture medium takes a Neurobasal-A culture medium as a basal culture medium, and further comprises: 1-3% of B-27 type serum-free additive, 0.2-2% of L-glutamine, 0.05-0.2% of GDNF and 1-5% of 100 multiplied antibacterial-antifungal agent.
The invention firstly takes bladder bodies, washes the bladder bodies by sterile Kirschner fluid and then cuts the bladder bodies into pieces to obtain a pretreated bladder sample. In the invention, the washing times of the sterile Kirschner liquid are preferably 2-3 times; in the invention, the sterile Kirschner liquid washing is preferably operated on ice in the whole process, and the Kirschner liquid is aerated with oxygen in advance for more than half an hour to reach the oxygen saturation in the liquid.
The reason for adopting sterile Kirschner fluid to wash is that the Kirschner fluid can provide necessary components required by the survival of fresh tissues, the pH is proper, the Kirschner fluid which is aerated in advance is in an oxygen saturation state and can provide oxygen, and other buffer solutions which meet the survival requirements of the tissues can also be adopted, but certain cleanliness is required. The invention has no special limitation on the source and the dosage of the Kirschner solution. In the present invention, the shearing is preferably performed by shearing the washed bladder body into small pieces having a diameter of 0.05cm or less.
After obtaining the pretreated bladder sample, the present invention pretreats the bladder sample with type II collagenase digestion, centrifuges and discards the supernatant to obtain a first digest. In the invention, the concentration of the type II collagenase is preferably 1-5 mg/ml, and more preferably 2 mg/ml; the collagenase of type II can be purchased from commercial products. In the invention, the ratio of the volume of the type II collagenase with the concentration of 1-5 mg/ml to the mass of the pretreated bladder sample is preferably 1-5 ml: 1g, more preferably 2-3 ml: 1g of the total weight of the composition. In the present invention, the digestion is preferably at 37 ℃ with 5% CO2And (5) digesting in a shaking table in an incubator. In the invention, the digestion time is preferably 1-2 min, and more preferably 1.5 min. In the invention, the centrifugal force is preferably 300-500 g, more preferably 350-450 g; the time for centrifugation is preferably 8-16 min, and more preferably 10-15 min.
After the first digestion product is obtained, the first digestion product is digested by trypsin for 3-10 min, and the supernatant is centrifuged and discarded to obtain a second digestion product. In the invention, the mass concentration of the trypsin is preferably 0.02-0.1%, and more preferably 0.05%; the trypsin of the present invention is purchased from a commercially available product. In the present invention, the ratio of the volume of trypsin at a mass concentration of 0.02 to 0.1% to the mass of the first digest is preferably 1 to 5 ml: 1g, more preferably 4 ml: 1g of the total weight of the composition. In the present invention, the time for digestion is preferably 5 min.
In the invention, when the trypsin digestion is stopped, the digestion is stopped by adding a buffer culture medium at 4 ℃; the buffer culture medium takes an F12 culture medium as a basic culture medium, and preferably comprises: fetal calf serum with the mass concentration of 5-12% and 100 multiplied by antibacterial-antifungal agent with the mass concentration of 0.5-3%; more preferably, it comprises: fetal bovine serum at a mass concentration of 10% and 100x antibacterial-antifungal agent at 0.1%. In the present invention, the 100 × antibacterial-antifungal agent is derived from commercially available products. In the present invention, the amount of the buffer medium added is preferably 2 times or more the total volume of the trypsin and the first digest.
And after obtaining the second digest, mixing the second digest with a neuron complete culture medium, filtering by a cell filter, carrying out shake culture on a filtrate for 20-40 min, centrifuging, removing a supernatant, resuspending the obtained precipitate with the neuron complete culture medium, adding the resuspension precipitate into a cell slide coated with poly-D-lysine and laminin in advance, culturing for 4-7D, carrying out full liquid change after 48h of culture, and carrying out full liquid change again after 48-96 h of culture. In the present invention, the time for the total volume change is determined based on the growth rate of the cells and the color change of the culture medium. In the present invention, the total replacement of the culture medium is to replace the whole culture medium with a new complete culture medium for neurons.
The neuron complete culture medium takes a Neurobasal-A culture medium as a basal culture medium, and preferably further comprises: 1-3% by mass of B-27 type serum-free additive, 0.2-2% by mass of L-glutamine, 0.05-0.2% by mass of GDNF and 1-5% by mass of 100x antibacterial-antifungal agent; more preferably, it comprises: 2% by mass of B-27 type serum-free additive, 0.1% by mass of L-glutamine, 0.1% by mass of GDNF and 3% by mass of 100 × antibacterial-antifungal agent. The complete neuron culture medium provided by the invention is a growth factor required by the growth of neurons.
In the present invention, it is preferable that the ratio of the mass of the pretreated bladder sample to the volume of the neuron complete medium is 1 g: mixing the second digest with a neuron complete medium at a ratio of 0.1-2 ml; the ratio is more preferably 1 g: 1 to 1.5 ml. In the present invention, the inner diameter of the cell filter is preferably 40 to 100 μm, and more preferably 70 μm. In the present invention, the digestion is preferably carried out in a water bath at 37 ℃.
In the present invention, the method for preparing the cell-climbing sheet pre-coated with poly-D-lysine and laminin preferably comprises the following steps: the polylysine was coated at a concentration of 0.1mg/ml for 10 minutes, followed by laminin at a concentration of 50. mu.g/ml for 1 hour.
In the present invention, the time for the shake culture is preferably 30 min. The shaking table culture is carried out after the filtration of the cell filter, so that cell clusters, tissue blocks and tissue fragments are dispersed under the action of the shaking table.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Taking out a rat bladder, rinsing the rat bladder for 3 times in sterile Kirschner solution, and shearing the rat bladder to be less than 0.05cm in diameter to obtain a pretreated bladder sample; adding 10ml and 2mg/ml type II collagenase into 1g pretreated bladder sample, and digesting for about 60min at 37 deg.C with 5% CO2Shaking table digestion in cell culture box, centrifuging at 356g for 8min, and discarding supernatant to obtain first digest. Mixing the first digest with 5ml of 0.05% trypsin solution, digesting in water bath at 37 ℃ for 5 minutes, immediately adding two times of 4 ℃ buffer medium to terminate digestion, centrifuging 356g for 8min, and discarding the supernatant to obtain a second digest.
The second digest was mixed with 3mL of complete neuronal medium and resuspended, and filtered through a 70 μm inner diameter cell filter to obtain a cell filtrate. The cell filtrate was cultured on a shaker at 20rpm for 30 minutes, 356g was centrifuged for 8min, the supernatant was discarded, and the resulting pellet was mixed with 1ml of complete medium and resuspended to obtain a cell resuspension solution. Adding cell suspension (400. mu.l per well of 48-well plate) to cell-slide coated with poly-D-lysine and laminin (poly-lysine concentration 0.1mg/ml is coated for 10min, laminin concentration 50 μ g/ml is coated for 1 hr), placing the culture plate at 37 deg.C, and 5% CO2Culturing in a cell culture box, fully changing the liquid after 48 hours, changing the liquid for the 2 nd time after 72 hours,then adding complete neuron culture medium to the original volume of 400 mu L to obtain primary neuron cells of rat bladder.
Buffer culture medium: f12 medium containing 10% Fetal Bovine Serum (FBS) and 1% antibiotic/antifungal agent.
Complete neuron culture medium: Neurobasal-A medium containing 2% B-27 serum-free additive, 1% L-glutamine, 1% fetal bovine serum, 0.1% glial cell-derived neurotrophic factor (GDNF), and 3% antibiotic/antifungal 100 Xliquid.
Example 2
The primary neuron cells of rat bladder separated and extracted according to the method shown in example 1 were examined by using an optical microscope and a confocal laser microscope LSM800, and the results are shown in fig. 1 to 4.
Fig. 1 and 2 are photographs of an optical microscope observed under a 200-fold microscope. FIGS. 3 and 4 are photographs of a laser confocal microscope LSM800 observed under a 20 Xobjective, in which blue fluorescence is hoechst 33342 (nucleus) and green fluorescence is β -III-tubulin (cytoskeletal protein of neuronal cells).
As can be seen from FIGS. 1 to 4, β -III-tublin is a marker for neuronal cells, and cells in the specific staining representation are neuronal cells.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A method for separating and extracting primary neurons of bladder comprises the following steps:
(1) taking a bladder body, washing the bladder body by sterile Kirschner liquid, and shearing the bladder body to obtain a pretreated bladder sample;
(2) digesting the pretreated bladder sample with type II collagenase, centrifuging and discarding the supernatant to obtain a first digest;
the concentration of the type II collagenase is 1-5 mg/ml, and the ratio of the volume of the type II collagenase to the mass of the pretreated bladder sample is 1-5 ml: 1g of a compound;
the digestion time of the type II collagenase is 60 min;
(3) digesting the first digestion product with trypsin for 3-7 min, centrifuging and removing a supernatant to obtain a second digestion product;
(4) mixing the second digest with a neuron complete culture medium, filtering by a cell filter, carrying out shake culture on a filtrate for 20-40 min, centrifuging, removing a supernatant, carrying out heavy suspension on the obtained precipitate by using the neuron complete culture medium, adding the heavy suspension to a cell slide coated with poly-D-lysine and laminin in advance, culturing for 4-7D, carrying out full liquid change after 48h of culture, and carrying out full liquid change again after 48-96 h of culture;
the neuron complete culture medium takes a Neurobasal-A culture medium as a basal culture medium, and further comprises: 1-3% of B-27 type serum-free additive, 0.2-2% of L-glutamine, 0.05-0.2% of GDNF and 1-5% of 100 multiplied antibacterial-antifungal agent.
2. The separation and extraction method according to claim 1, wherein in the step (3), when the trypsin digestion is terminated, the digestion is terminated by adding a buffer medium at 4 ℃;
the buffer culture medium takes an F12 culture medium as a basic culture medium, and further comprises: fetal calf serum with the mass concentration of 5-12% and 100 multiplied by antibacterial-antifungal agent with the mass concentration of 0.5-3%.
3. The method of claim 2, wherein the amount of the buffer medium added in step (3) is 2 times or more the total volume of the trypsin and the first digest.
4. The separation and extraction method according to claim 1, wherein the inner diameter of the cell filter is 40 to 100 μm.
5. The separation and extraction method according to claim 1, wherein in the step (2), the digestion is carried out at 37 ℃5％CO2And (5) digesting in a shaking table in an incubator.
6. The separation and extraction method according to claim 1, wherein in the step (3), the ratio of the mass of the pretreated bladder sample to the volume of the neuron complete culture medium is 1 g: mixing the second digest with a neuron complete medium at a ratio of 0.1-2 ml.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274055A (en) * | 2015-06-19 | 2016-01-27 | 南通大学 | Neuron primary culture purification method |
| CN106867965A (en) * | 2017-04-18 | 2017-06-20 | 南京盖斯夫医药科技有限公司 | A kind of efficient differentiation-inducing agents of fat stem cell and inductive differentiation medium |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105274055A (en) * | 2015-06-19 | 2016-01-27 | 南通大学 | Neuron primary culture purification method |
| CN106867965A (en) * | 2017-04-18 | 2017-06-20 | 南京盖斯夫医药科技有限公司 | A kind of efficient differentiation-inducing agents of fat stem cell and inductive differentiation medium |
Non-Patent Citations (3)
| Title |
|---|
| Isolation and Culture of Primary Neurons and Glia From Adult Rat Urinary Bladder;HongYing Cao,et al;《J Vis Exp》;20200523;全文 * |
| Time‐dependent functional, morphological, and molecular changes in diabetic bladder dysfunction in streptozotocin‐induced diabetic mice;Hongying Cao，et al;《Neurourol Urodyn》;20190420;全文 * |
| 肾上腺素 β3 受体调节大鼠逼尿肌舒张的机制;曹文锋等;《四川医学》;20120331;全文 * |
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