CN111808794A

CN111808794A - Method for efficiently obtaining primary organ cells of mice

Info

Publication number: CN111808794A
Application number: CN202010588427.3A
Authority: CN
Inventors: 宋海长; 朱剑军; 马荣林
Original assignee: Jiangxi Boling Biotechnology Co ltd
Current assignee: Jiangxi Boling Biotechnology Co ltd
Priority date: 2020-06-24
Filing date: 2020-06-24
Publication date: 2020-10-23
Anticipated expiration: 2040-06-24
Also published as: CN111808794B

Abstract

The invention discloses a method for efficiently obtaining primary organ cells of a mouse, and belongs to the technical field of cell culture. The invention adopts a separation method combining mechanical separation and enzyme digestion, a tissue dissociation reagent prepared by a tissue dissociation kit can fully digest organ tissues of mice, the damage to cells is minimized, and a powerful guarantee is provided for the separation and acquisition of primary organ cells, and the obtained primary organ cells are observed and identified by cell morphology: the number is large, the activity is high, and the initial survival rate of cells is more than 85%. The method provided by the invention is economical, practical, simple and feasible, is favorable for establishing in vitro experiment model cells, researching the characteristics of organ cells and providing reliable cell resources for subsequent experiments.

Description

Method for efficiently obtaining primary organ cells of mice

Technical Field

The invention relates to a method for efficiently obtaining primary organ cells of a mouse, belonging to the technical field of cell culture.

Background

With the rapid development of modern technology, the research of using mice as human disease model animals is more and more common. The maintenance and development of normal physiological state of mouse, which is a common human disease model animal, is inseparable from the division of work and cooperation of its main tissues and organs. Tissues and organs of normal mice include: liver, kidney, lung, heart, spleen, stomach, intestine, etc. Different tissues, organs, play different roles in the normal life activities of mice. For example, the liver of reddish brown is an organ mainly having a metabolic function, and its main functions are to secrete bile, store glycogen, regulate metabolism of protein, fat and carbohydrate, etc., and also to detoxify, hematopoiesis and coagulate blood; the kidney of the hyacinth bean shape, its basic function is to produce urine, in order to clear up metabolic product and some wastes, poison in vivo, keep moisture and other useful substances through the function of reabsorption at the same time, guarantee the stability of the internal environment of organism, make metabolism go on normally; the lung, which is the respiratory organ of the body and also the important hematopoietic organ of the body, has the basic functions of respiration, continuously inhaling clear air, discharging foul air, inhaling refreshment, realizing the gas exchange between the body and the external environment so as to maintain the life activities of the body; the heart, which is composed of cardiac muscle, functions to promote blood flow, provide sufficient blood flow to organs and tissues to supply oxygen and various nutrients, and remove metabolic end products (such as carbon dioxide, inorganic salts, urea, uric acid, etc.), so that cells maintain normal metabolism and function. Therefore, the method has important significance in the aspects of researching medicaments related to tissues and organs and action mechanisms by culturing mouse tissues and organ cells in vitro and detecting the functions of the mouse tissues and organ cells. In vitro culture of tissue and organ cells is also a prerequisite and basis for the study of their morphology, structure and function.

In the prior art, the method for obtaining the mouse primary tissue and the mouse primary organ cells mainly comprises a portal vein intubation in-situ or in-vitro perfusion combined collagenase digestion method, an in-vitro two-step perfusion method, a shear digestion method and the like, the methods are complex to operate, the separation and culture technologies are greatly different, the activity and the quantity of the obtained primary tissue and the primary organ cells are greatly different, and meanwhile, the cell characteristics such as cell surface epitope damage and the like exist, so that the cells are influenced to be used for subsequent experiments.

Disclosure of Invention

In order to solve the problems, the invention provides a method for efficiently obtaining the primary organ cells of the mouse, which has simple operation and high cell number and survival rate, is an ideal method for obtaining the primary organ cells of the mouse, and can meet the requirements of various physiological and biochemical experiments.

The first purpose of the invention is to provide a method for efficiently obtaining mouse primary organ cells, which comprises the following steps:

s1, taking fresh mouse organ tissues, cutting the fresh mouse organ tissues into small pieces, adding a precooling buffer solution for cleaning, and centrifuging to remove a supernatant;

s2, resuspending the tissue by using a preheated tissue dissociation reagent, and processing the organ tissue by using a tissue crushing cell separator after resuspension;

s3, placing the tissue resuspension solution processed in the step S2 at 35-38 ℃ and 100-300 rpm, and digesting the organ tissue for 10-100 min;

s4, after digestion, processing the organ tissues by adopting a tissue disruption cell separator to obtain tissue homogenate;

s5, filtering the tissue homogenate, collecting filtrate, and centrifuging to remove supernatant;

s6, adopting buffer solution for heavy suspension, adding erythrocyte lysate to remove erythrocytes, and centrifuging to remove supernatant;

s7, washing the cells by adopting a buffer solution to obtain the primary organ cells of the mouse;

the tissue dissociation reagent is: 0.2-0.4 mg/mL hyaluronidase, 0.1-0.2 mg/mL DNase I, 1-5 mg/mL collagenase, 5-15% serum and 0.5-2% double antibody cell culture medium.

Further, the collagenase comprises collagenase D, collagenase type I or collagenase type IV.

Further, the primary organ cell is one of liver cell, lung cell, kidney cell and heart cell.

Further, when the primary organ cells are liver cells, the cell culture medium is DMEM containing 2-4 mg of EDTA, and when the primary organ cells are lung cells, kidney cells or heart cells, the cell culture medium is RPMI-1640.

Further, the mice are 8-15 weeks C57BL/6J mice.

Further, in step S1, the organ tissues of the fresh mouse are cut into pieces of 2-5 mm.

Further, in the step S1, the pre-cooling buffer is pre-cooled at 0-4 ℃.

Further, the preheated tissue dissociation reagent is preheated to 35-38 ℃.

Further, the centrifugation condition is 400 Xg, and 5-10 min.

Further, the erythrocyte lysate comprises ammonium chloride and tris (hydroxymethyl) aminomethane.

Further, the double-resistant agent is penicillin 50-150 u/mL and streptomycin 50-150 u/mL.

The second purpose of the invention is to provide the mouse primary organ cells prepared by the method.

The invention has the beneficial effects that:

the invention adopts a separation method combining mechanical separation and enzyme digestion, a tissue dissociation reagent prepared by a tissue dissociation kit can fully digest organ tissues of mice, the damage to cells is minimized, and a powerful guarantee is provided for the separation and acquisition of primary organ cells, and the obtained primary organ cells are observed and identified by cell morphology: high cell activity, high cell survival rate over 85%. The method provided by the invention is economical, practical, simple and feasible, is favorable for establishing in vitro experiment model cells, researching the characteristics of organ cells and providing reliable cell resources for subsequent experiments.

Drawings

FIG. 1 shows the activity of liver cells detected by trypan blue staining method at different culture times;

FIG. 2 is a picture of mouse hepatocytes cultured for 0-48 h, 10 ×;

FIG. 3 shows the activity of lung cells detected by trypan blue staining method at different culture times;

FIG. 4 is a picture of lung cells of mice cultured for 0-48 h, 10 ×;

FIG. 5 shows the activity of kidney cells measured by trypan blue staining for different culture times;

FIG. 6 is a picture of kidney cells of mice cultured for 0-48 h, 10 ×;

FIG. 7 shows the activity of heart cells detected by trypan blue staining for different culture times;

FIG. 8 is a photograph of cardiac cells of mice cultured for 0-48 h, 10 ×;

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Tissue disruption cell separator: see patent CN201920276309.1 separator for living cells in biological tissue.

Example 1: mouse hepatocyte harvesting

(1) Placing the tissue processing apparatus in an aluminum box with a cover, wrapping the aluminum box with kraft paper, placing in a high pressure steam sterilization pot, sterilizing at 121 deg.C for 20min, taking out the apparatus, and air drying in a biological safety cabinet;

(2) selecting a C57BL/6J mouse with the age of 8-15 weeks, anesthetizing with chloral hydrate, soaking in 75% ethanol water solution for 2min, fixing the mouse on a plate with the abdominal surface facing upwards, sterilizing the abdomen with 75% ethanol, cutting the midline of two legs to expose the abdominal cavity, allowing red-brown liver tissues to be seen, separating the liver tissues with surgical scissors, and placing in a culture dish;

(3) transferring the liver tissue to a centrifuge tube, shearing the liver tissue into small pieces of 2-5 mm by using surgical scissors, adding a proper amount of precooled PBS under an aseptic condition, slightly blowing and beating the small pieces by using a pipette gun, placing the small pieces in a centrifuge for centrifugation, and discarding supernatant;

(4) resuspending the tissue by using 1mL of tissue dissociation reagent prepared by a tissue dissociation kit preheated at 37 ℃, pouring the tissue into a C tube, washing a centrifuge tube by using 1mL of the same tissue dissociation reagent preheated at 37 ℃ and pouring the same tissue dissociation reagent into the C tube, adding 3mL of the same tissue dissociation reagent preheated at 37 ℃ into the C tube, and screwing down a tube cover of the C tube;

the tissue dissociation reagent prepared by the tissue dissociation kit comprises: 10mg/mL hyaluronidase, 5mg/mL DNase I, and 20mg/mL collagenase D were pipetted into 100uL, and 250uL of the above-mentioned reagents, respectively, and 4.55mL DMEM containing 10% serum and 1% diabody (100u/mL penicillin, 100u/mL streptomycin) was added thereto, and 2mg EDTA was added thereto and mixed well.

(5) Inverting the tube C on a tissue disruption cell separator, starting an instrument program, and treating liver tissues;

(6) placing the tube C in a constant temperature shaking table at 37 ℃ to digest the liver tissue;

(7) taking out the tube C from the constant temperature shaking table, inverting the tube C on the tissue broken cell separator, starting an instrument program, and treating the liver tissue;

(8) filtering the tissue homogenate in the tube C, collecting filtrate by using a 50mL centrifuge tube, placing the filtrate in a centrifuge for centrifugation, and discarding supernatant;

(9) resuspending with PBS, removing erythrocytes with erythrocyte lysate, centrifuging with centrifuge, discarding supernatant, resuspending with PBS, centrifuging, discarding supernatant, and clarifying the resuspension solution;

(10) and (3) observing cell morphology and growth conditions: resuspending the cells with a proper amount of PBS, and observing the shape and growth condition of the primarily cultured mouse hepatocytes by using an inverted microscope;

(11) taking a proper amount of cell suspension for trypan blue staining, sucking 10uL of the stained cell suspension, slowly pushing the cell suspension along the edge of an upper cover plate of a cell counting plate until the suspension just fills the lower part of the cover plate, observing under an inverted microscope, indicating that liver cells survive if the cells are not stained, indicating that the cells die if the cells are stained into blue, and counting the number of cells in four lattices (the total number of the cells in four lattices/4 multiplied by 10)⁴) And calculating the cell survival rate: (total number of cells-total number of dead cells)/total number of cells × 100%, the results are shown in tables 1 and 2 below:

TABLE 1

TABLE 2

The mouse obtained by the invention has a large number of liver cells and high cell survival rate.

Example 2: trypan blue staining method for detecting activity of liver cells in different culture times

Primary hepatocytes of mice were obtained according to the method of steps (1) to (9) of example 1, and the cells were resuspended in an appropriate amount of DMEM containing 10% fetal bovine serum and 1% double antibody, transferred to a culture dish, and placed at 37 ℃ in 5% CO₂Culturing in an incubator; when the culture time was 0h, 12h, 24h, and 48h, respectively, the cells were stained with trypan blue according to the method in step (11), and the cell viability was calculated, as shown in table 3 and fig. 1, and the morphology and growth status of the cells during the culture are shown in fig. 2.

TABLE 3

Comparative example 1:

tissue dissociation reagent prepared by mixing collagenase type IV with the concentration of 500uL and 5mg/mL with DMEM containing 10% of serum and 1% of double antibody (penicillin 100u/mL and streptomycin 100 u/mL) is adopted to dissociate tissues instead of the tissue dissociation reagent prepared by the tissue dissociation kit, other conditions are consistent with those of example 1, the obtained primary mouse liver cells are re-suspended by using a proper amount of PBS, the shape and growth condition of the primary cultured mouse liver cells are observed by using an inverted microscope, and the results of cell viability detection by a trypan blue staining method are shown in Table 4.

Comparative example 2:

the hyaluronidase with the concentration of 10mg/mL, DNase I with the concentration of 5mg/mL and collagenase D with the concentration of 20mg/mL are adopted, 50uL and 250uL of the reagents are respectively absorbed, 4.65mL of DMEM containing 10% serum and 1% double antibody (100u/mL penicillin and 100u/mL streptomycin) is added, 1mg of EDTA is added and mixed, the tissue is dissociated by replacing a tissue dissociation reagent prepared by a tissue dissociation kit, other conditions are consistent with those of example 1, the obtained primary mouse liver cells are resuspended by using a proper amount of PBS, the morphology and the growth condition of the mouse liver cells after being cultured are observed by an inverted microscope, and the results of detecting the cell viability by a trypan blue staining method are shown in Table 4.

TABLE 4

Example 3: mouse lung cell harvest

(2) selecting a C57BL/6J mouse with the age of 8-15 weeks, anesthetizing with chloral hydrate, soaking in 75% ethanol water solution for 2min, fixing the mouse on a plate with the abdominal surface facing upwards, sterilizing the abdomen with ethanol, cutting the midline of two legs to expose the abdominal cavity and the thoracic cavity, showing lung tissues similar to a cavernous body and a left lung tissue and a right lung tissue, inserting an injector which absorbs PBS into the bottom of the heart, injecting PBS to wash the lung to white, separating the lung tissues by surgical scissors, and placing the lung tissues in a culture dish;

(3) transferring lung tissues to a centrifuge tube, shearing the lung tissues into small pieces of 2-5 mm by using surgical scissors, adding a proper amount of precooled PBS under aseptic conditions, slightly blowing and beating the pieces by using a pipette gun, placing the pieces in a centrifuge for centrifugation, and discarding supernatant;

(4) resuspending the tissue by using 1mL of tissue dissociation reagent prepared by a tissue dissociation kit preheated at 37 ℃, pouring the tissue into a C tube, flushing a centrifuge tube by using 1mL of the same tissue dissociation reagent preheated at 37 ℃ and pouring the same tissue dissociation reagent into the C tube, adding 3mL of the same tissue dissociation reagent preheated at 37 ℃ into the C tube, and screwing down a tube cover of the C tube;

the tissue dissociation reagents prepared in the tissue dissociation kit were 10mg/mL hyaluronidase, 5mg/mL DNAse I, and 50mg/mL collagenase type I, and 100uL, and 500uL of the above reagents were pipetted and added to 4.30mL RPMI-1640 containing 10% serum and 1% diabody (100u/mL penicillin, 100u/mL streptomycin), respectively, and mixed well.

(5) Inverting the tube C on a tissue broken cell separator, starting an instrument program, and treating lung tissues;

(6) placing the tube C in a constant temperature shaking table with the temperature of 37 ℃ and the rpm of 200 to digest the lung tissue;

(7) taking out the tube C from the constant-temperature shaking table, inverting the tube C on the tissue broken cell separator, starting an instrument program, and treating lung tissues;

(10) and (3) observing cell morphology and growth conditions: resuspending the cells with a proper amount of PBS, and observing the shape and growth condition of the primarily cultured mouse lung cells by using an inverted microscope;

(11) taking a proper amount of cell suspension obtained in the step (10), carrying out trypan blue staining, sucking 10uL of the stained cell suspension, slowly pushing the cell suspension along the edge of a cover plate on a cell counting plate until the lower part of the cover plate is just filled with the suspension, observing under an inverted microscope, indicating that lung cells survive if the cells are not stained, indicating that the cells die if the cells are stained into blue, and counting the number of cells in four lattices (the total number of the cells in four lattices/4 multiplied by 10)⁴) And calculating the cell survival rate: (total number of cells-total number of dead cells)/total number of cells × 100%, the results are shown in tables 5 and 6 below:

TABLE 5

TABLE 6

Example 4: trypan blue staining method for detecting activity of lung cells at different culture times

Primary mouse lung cells were obtained according to the method of steps (1) to (9) of example 1, resuspended in an appropriate amount of 10% fetal bovine serum and 1% double antibody-containing RPMI-1640, transferred to a petri dish, and placed at 37 ℃ in 5% CO₂Culture ofCulturing in a culture box; when the culture time was 0h, 12h, 24h, and 48h, respectively, the cells were stained with trypan blue according to the method in step (11), and the cell viability was calculated, as shown in table 7 and fig. 3, and the cell morphology and growth state during the culture were as shown in fig. 4.

TABLE 7

Comparative example 3:

tissue dissociation reagent prepared by sucking 625uL of collagenase type I with the concentration of 40mg/mL and 4.375mL of RPMI-1640 containing 10% serum and 1% double antibody (100u/mL penicillin and 100u/mL streptomycin) was used instead of the tissue dissociation reagent prepared by the tissue dissociation kit to dissociate tissue, the other conditions were the same as those in example 1, the obtained primary mouse lung cells were resuspended in an appropriate amount of PBS, the morphology and growth status of the mouse lung cells after primary culture were observed by an inverted microscope, and the results of cell viability detection by trypan blue staining method are shown in Table 8.

Comparative example 4:

the results of detecting cell viability by trypan blue staining are shown in Table 8, wherein hyaluronidase at a concentration of 10mg/mL, DNase I at a concentration of 5mg/mL, and collagenase D at a concentration of 20mg/mL are used, 50uL, and 250uL of the above reagents are aspirated separately, 4.65mL of RPMI-1640 containing 10% serum and 1% diabody (100u/mL penicillin, 100u/mL streptomycin) is added, tissue dissociation reagent prepared in a tissue dissociation kit is replaced, other conditions are the same as those in example 1, the obtained primary mouse lung cells are resuspended in an appropriate amount of PBS, morphology and growth status of the primary mouse lung cells after culture are observed by an inverted microscope.

TABLE 8

Example 5: mouse renal cell harvesting

(2) selecting a C57BL/6J mouse with the age of 8-15 weeks, anesthetizing with chloral hydrate, soaking in 75% ethanol water solution for 2min, fixing the mouse on a plate with the abdominal surface facing upwards, sterilizing the abdomen with 75% ethanol, cutting the midline of two legs to expose the abdominal cavity, allowing a hyacinth bean-shaped and reddish brown kidney tissue to be visible, separating the kidney tissue with surgical scissors, and placing the kidney tissue in a culture dish;

(3) transferring the kidney tissues to a centrifuge tube, shearing the kidney tissues into small pieces of 2-5 mm by using surgical scissors, adding a proper amount of precooled PBS under an aseptic condition, slightly blowing and beating the kidney tissues by using a pipette gun, placing the kidney tissues in a centrifuge for centrifugation, and discarding the supernatant;

(5) Inverting the tube C on a tissue disruption cell separator, starting an instrument program, and treating kidney tissues;

(6) placing the tube C in a constant temperature shaking table at 37 ℃ to digest kidney tissues;

(7) taking out the tube C from the constant temperature shaking table, inverting the tube C on the tissue disruption cell separator, starting an instrument program, and treating kidney tissues;

(10) and (3) observing cell morphology and growth conditions: resuspending the cells with a proper amount of PBS, and observing the shape and growth condition of the primarily cultured mouse kidney cells by using an inverted microscope;

(11) taking a proper amount of cell suspension obtained in the step (10), carrying out trypan blue staining, sucking 10uL of the stained cell suspension, slowly pushing the cell suspension along the edge of a cover plate on a cell counting plate until the lower part of the cover plate is just filled with the suspension, observing under an inverted microscope, indicating that kidney cells survive if the cells are not stained, indicating that the cells die if the cells are stained into blue, and counting the number of cells in four lattices (the total number of the cells in four lattices/4 multiplied by 10)⁴) And calculating the cell survival rate: (total number of cells-total number of dead cells)/total number of cells × 100%, the results are shown in tables 9 and 10 below:

TABLE 9

Watch 10

Example 6: trypan blue staining method for detecting activity of kidney cells at different culture times

Mouse primary kidney cells were obtained according to the method of steps (1) to (9) of example 1, resuspended in an appropriate amount of RPMI-1640 containing 10% fetal bovine serum and 1% double antibody, transferred to a petri dish, and placed at 37 ℃ in 5% CO₂Culturing in an incubator; when the culture time was 0h, 12h, 24h, and 48h, respectively, the cells were stained with trypan blue according to the method in step (11), and the cell viability was calculated, as shown in table 11 and fig. 5, and the cell morphology and growth state during the culture were as shown in fig. 6.

TABLE 11

Comparative example 5:

tissue dissociation reagent prepared by sucking 625uL of collagenase type IV with concentration of 8mg/mL and 4.375mL of RPMI-1640 containing 10% serum and 1% double antibody (100u/mL penicillin, 100u/mL streptomycin) was used instead of the tissue dissociation reagent prepared by the tissue dissociation kit to dissociate tissue, the other conditions were the same as those in example 1, the obtained primary mouse kidney cells were resuspended in an appropriate amount of PBS, morphology and growth status of the primary mouse kidney cells after culture were observed with an inverted microscope, and the results of cell viability detection by trypan blue staining method are shown in Table 12.

Comparative example 6:

the results of measuring cell viability by trypan blue staining method are shown in Table 12, wherein hyaluronidase with a concentration of 10mg/mL, DNase I with a concentration of 5mg/mL, and collagenase D with a concentration of 20mg/mL are used, 50uL, and 250uL of the above reagents are aspirated separately, 4.65mL of RPMI-1640 containing 10% serum and 1% diabody (100u/mL penicillin, 100u/mL streptomycin) is added, tissue dissociation reagent prepared in a tissue dissociation kit is replaced, other conditions are the same as those in example 1, the obtained primary mouse kidney cells are resuspended in an appropriate amount of PBS, morphology and growth status of the mouse kidney cells after primary culture are observed by an inverted microscope.

TABLE 12

Example 7: mouse cardiac cell harvesting

(2) selecting a C57BL/6J mouse with the age of 8-15 weeks, anesthetizing with chloral hydrate, soaking in 75% ethanol water solution for 2min, fixing the mouse on a plate with the abdominal surface facing upwards, disinfecting the abdominal part with ethanol, cutting the midline of two legs to expose the abdominal cavity and the thoracic cavity, separating the heart tissue from the heart tissue, and placing the heart tissue in a culture dish;

(3) transferring the heart tissue to a centrifuge tube, shearing the heart tissue into small pieces of 2-5 mm by using surgical scissors, adding a proper amount of precooled PBS under an aseptic condition, slightly blowing and beating the small pieces by using a pipette gun, placing the small pieces in a centrifuge for centrifugation, and discarding supernatant;

(4) resuspending the tissue by 1mL of a tissue dissociation reagent which is preheated at 37 ℃ and prepared by a tissue dissociation kit, pouring the tissue into a C tube, flushing a centrifuge tube by 1mL of the same tissue dissociation reagent which is preheated at 37 ℃ and pouring into the C tube, adding 3mL of the same tissue dissociation reagent which is preheated at 37 ℃ into the C tube, and screwing down a tube cover of the C tube;

the tissue dissociation reagents prepared by the tissue dissociation kit are 10mg/mL hyaluronidase, 5mg/mL DNase I and 20mg/mL collagenase type IV, 100uL and 250uL of the above reagents are respectively absorbed and added into 4.55mL RPMI-1640 containing 10% serum and 1% double antibody (100u/mL penicillin and 100u/mL streptomycin), and the mixture is fully mixed.

(5) Inverting the tube C on a tissue broken cell separator, starting an instrument program, and treating the heart tissue;

(6) placing the tube C in a constant temperature shaking table at 37 ℃ and 200rpm, and digesting cardiac tissues;

(7) taking out the tube C from the constant temperature shaking table, inverting the tube C on the tissue broken cell separator, starting an instrument program, and treating the heart tissue;

(10) and (3) observing cell morphology and growth conditions: resuspending the cells with a proper amount of PBS, and observing the shape and growth condition of the primarily cultured mouse heart cells by using an inverted microscope;

(11) taking a proper amount of cell suspension obtained in the step (10), carrying out trypan blue staining, sucking 10uL of the stained cell suspension, slowly pushing the cell suspension along the edge of a cover plate on a cell counting plate until the lower part of the cover plate is just filled with the suspension, observing under an inverted microscope, indicating that the heart cells survive if the cells are not stained, and indicating that the heart cells survive if the cells are stained into blueIndicating cell death, the number of cells in the four large lattices (total number of cells in four large lattices/4X 10) was counted⁴) And calculating the cell survival rate: (total number of cells-total number of dead cells)/total number of cells × 100%, the results are shown in tables 13 and 14 below:

watch 13

TABLE 14

Example 8: trypan blue staining method for detecting activity of heart cells at different culture times

Primary mouse heart cells were obtained according to the methods of steps (1) to (9) of example 1, resuspended in an appropriate amount of 10% fetal bovine serum and 1% double antibody in RPMI-1640, transferred to a petri dish, and placed at 37 ℃ in 5% CO₂Culturing in an incubator; when the culture time was 0h, 12h, 24h, and 48h, respectively, the cells were stained with trypan blue according to the method in step (11), and the cell viability was calculated, as shown in table 15 and fig. 7, and the cell morphology and growth state during the culture were as shown in fig. 8.

Watch 15

Comparative example 7:

tissue dissociation reagent prepared by sucking 500uL of collagenase type I with a concentration of 50mg/mL and 4.50mL of RPMI-1640 containing 10% serum and 1% double antibody (100u/mL penicillin, 100u/mL streptomycin) was used instead of the tissue dissociation reagent prepared in the tissue dissociation kit to dissociate tissue, and the other conditions were the same as those in example 1, and the obtained primary mouse heart cells were resuspended in an appropriate amount of PBS, morphology and growth status of the mouse heart cells after primary culture were observed with an inverted microscope, and the results of cell viability detection by trypan blue staining method are shown in Table 16.

Comparative example 8:

the results of detecting cell viability by trypan blue staining are shown in Table 16, wherein hyaluronidase at a concentration of 10mg/mL, DNase I at a concentration of 5mg/mL, and collagenase D at a concentration of 20mg/mL are used, 50uL, and 250uL of the above reagents are aspirated separately, 4.65mL of RPMI-1640 containing 10% serum and 1% diabase (100u/mL penicillin, 100u/mL streptomycin) is added, tissue dissociation reagent prepared in a tissue dissociation kit is replaced, other conditions are the same as those in example 1, the obtained primary mouse heart cells are resuspended in an appropriate amount of PBS, morphology and growth status of the primary mouse heart cells after culture are observed by an inverted microscope.

TABLE 16

The results show that the method of the invention has the advantages of obviously more extracted cells, higher cell survival rate and cell activity.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

Claims

1. A method for efficiently obtaining primary organ cells of a mouse is characterized by comprising the following steps:

2. The method of claim 1, wherein the collagenase comprises collagenase type D, collagenase type i, or collagenase type iv.

3. The method of claim 1, wherein said primary organ cells are selected from the group consisting of hepatocytes, lung cells, kidney cells, and heart cells.

4. The method according to claim 3, wherein the cell culture medium is DMEM containing 2-4 mg EDTA when the primary organ cells are liver cells, and RPMI-1640 when the primary organ cells are lung cells, kidney cells or heart cells.

5. The method of claim 1, wherein the mouse is an 8-15 week C57BL/6J mouse.

6. The method according to claim 1, wherein in step S1, the pre-cooled buffer is pre-cooled at 0-4 ℃.

7. The method of claim 1, wherein the pre-heated tissue dissociation reagent is pre-heated to 35-38 ℃.

8. The method of claim 1, wherein the centrifugation is performed at 400 Xg for 5-10 min.

9. The method of claim 1, wherein the red blood cell lysate comprises ammonium chloride and tris.

10. The method as claimed in claim 1, wherein the diabase is penicillin 50-150 u/mL and streptomycin 50-150 u/mL.