CN116103224A - Preparation method of pig lung tissue suspension - Google Patents

Abstract

The invention discloses a preparation method of a pig lung tissue suspension, which comprises the following steps: obtaining a pig lung sample containing lung and trachea; injecting a culture medium into a lung main air pipe of the pig lung sample to obtain pig lung filled with the culture medium; using elbow pointed forceps to scratch and strip the pig lung filled with the culture medium to obtain a lung tissue block; mashing and filtering the lung tissue blocks to obtain lung tissue mashing liquid; homogenizing the lung tissue triturating liquid to obtain the pig lung tissue suspension. The preparation method of the pig lung tissue suspension provided by the invention is simple, efficient, easy to operate, time-saving and labor-saving, and can well avoid pollution and improve the cell preparation amount.

Description

Preparation method of pig lung tissue suspension

Technical Field

The invention relates to the technical field of biological tissue preparation, in particular to a preparation method of a pig lung tissue suspension.

Background

Rapid isolation and diagnosis of pathogenic microorganisms, particularly isolation, identification, culture, and the like of new viruses or strains, are often achieved by preparing a tissue suspension from a target tissue. The preservation conditions of the tissues in the transportation process, the selection of proper culture medium types during the preparation of the tissue suspension, the avoidance of pollution, the reduction of the introduction of non-target tissues, the shortening of the preparation time and the like are important conditions for efficiently preparing the tissue suspension and improving the preparation amount. There are many methods for diagnosing laboratory epidemic diseases, but the isolation of pathogenic microorganisms is one of the direct and efficient means. The target tissue is a disease material collected from the diseased animal body, provides a material for efficient preparation of tissue suspension and separation of pathogenic microorganisms, and provides a foundation for biological characteristic research of the separated microorganisms. The biological characteristics of pathogenic microorganisms have important guiding significance for preventing and treating epidemic diseases, ensure the scientific and effective prevention and control, reduce economic loss and play an important role in public health.

The lung is a respiratory organ in the body, and is divided into multiple lobes, and the number and size of lobes are different due to species differences. The air inside and outside the body enters alveoli through all levels of air pipes in the lung to be continuously exchanged, so that the normal operation of life is ensured. The bronchi are repeatedly branched into countless bronchioles, the tail ends of the bronchi are expanded into sacs, and the periphery of each sac is provided with a plurality of protruding vesicles, namely alveoli. Alveoli are the main sites of pulmonary gas exchange and are also functional units of the lung. Oxygen can reach alveoli through all levels of air pipes, diffuse from a high-permeability respiratory membrane to blood, realize gas exchange, and meanwhile, microorganisms can also reach the alveoli to cause immune response of the lung, eliminate invasive microorganisms and keep the body healthy. Pathogenic microorganisms are not necessarily destroyed, and can cause infectious respiratory diseases, cough and asthma when the respiratory diseases are mild; the weight affects the normal physiological functions of the organism and even dies. In animal husbandry, respiratory diseases have been seriously jeopardized the farming industry. The amount of pig breeding in China is the first in the world, and respiratory diseases cause huge economic losses, such as: the mycoplasma hyopneumoniae infection is followed by a chronic respiratory infectious disease with high morbidity and low mortality, and only infected pigs are currently found, and the pigs are not classified into age, sex and breed. The biological characteristic research of the pathogenic bacteria can be realized by preparing lung tissue suspension for infecting pigs, so that the vaccine aiming at the pathogenic bacteria is prepared, and has been verified and approved by the market.

The conventional preparation method of the pig lung tissue suspension mainly adopts mechanical methods, such as a shearing method and a grinding method, can realize separation of cells or microorganisms, and is mainly used for laboratory researches, but the conventional preparation method mainly has the following problems that pollution exists in the preparation process, and non-target tissues such as: connective tissue and the like are easy to be mixed in tissue suspension, the preparation amount is unstable, the preparation amount is small, the operation time is too long, pollution is difficult to control, and the like.

Disclosure of Invention

The invention mainly aims to provide a preparation method of a pig lung tissue suspension, and aims to solve the problems of easiness in pollution and small preparation amount of the existing preparation method of the pig lung tissue suspension.

In order to achieve the above purpose, the invention provides a preparation method of a pig lung tissue suspension, which comprises the following steps:

obtaining a pig lung sample containing lung and trachea;

injecting a culture medium into a lung main air pipe of the pig lung sample to obtain pig lung filled with the culture medium;

using elbow pointed forceps to scratch and strip the pig lung filled with the culture medium to obtain a lung tissue block;

mashing and filtering the lung tissue blocks to obtain lung tissue mashing liquid;

homogenizing the lung tissue triturating liquid to obtain the pig lung tissue suspension.

Optionally, in the step of obtaining a pig lung sample containing lung and trachea: the main air pipe is clamped by a rope, and the sample is sealed and refrigerated for transportation.

Optionally, in the step of injecting the culture medium into the lung main trachea of the pig lung sample, injecting the culture medium into the lung main trachea of the pig lung sample using an input drainage device, the input drainage device comprising:

the input system comprises a first container, a perfusion tube and a pump, wherein the first container is used for containing the culture medium, one end of the perfusion tube is communicated into the first container, the other end of the perfusion tube is provided with a stomach-filling needle head which is used for being inserted into the pig lung sample, the perfusion tube is connected to a power clamping groove of the pump so as to pump the culture medium in the first container through the pump and convey the culture medium into the pig lung sample, and the pump is provided with a controller which is used for starting or stopping transfusion and controlling the flow rate of the transfusion; the method comprises the steps of,

the condensing system comprises a second container and a thermometer, wherein the second container is used for containing an ice-water mixture and the first container, so that the first container is in an ice-water mixture environment, and the thermometer is arranged in the second container and used for detecting the temperature in the second container.

Optionally, the step of injecting the culture medium into the lung main trachea of the pig lung sample using an input drainage device:

and (3) injecting culture medium into the lung main air pipe of the pig lung sample at an initial flow rate of 150-250 mL/min until the lung filling degree reaches 80-90%, regulating the infusion flow rate to 15-25 mL/min until the lung is completely filled, stopping injecting the culture medium, taking out the needle head and clamping the lung main air pipe.

Optionally, the stomach irrigation needle is a 16-20-size elbow stomach irrigation needle.

Optionally, the step of injecting a culture medium into the lung main trachea of the pig lung sample to obtain a culture medium-filled pig lung:

the amount of the culture medium injected into each 100g of the pig lung sample is 500-1500 mL.

Optionally, the step of using the bent pointed forceps to scratch and strip the pig lung filled with the culture medium to obtain a lung tissue block comprises the following steps:

placing the pig lung filled with the culture medium in a shallow mouth container, pricking lung tissues with the elbow of the elbow pointed forceps, tearing the lung tissues along the parallel direction of the main air pipe of the lung, repeating the operation of tearing and stripping the lung tissues, so that each lung lobe tissue is torn into independent small lung tissues separated from the air pipe, and simultaneously, stripping the lung tissue blocks attached to the air pipe one by using the elbow parts of the pointed forceps, thereby obtaining the lung tissue blocks.

Optionally, in the step of mashing and filtering the lung tissue mass to obtain a lung tissue mashing solution, the step of mashing the lung tissue mass includes:

the lung tissue is circularly smashed by using a stainless steel smashing machine in a mode of smashing at a low speed and smashing at a high speed, wherein the rotating speed of the smashing at the high speed is 20000-25000 rpm, the single smashing time is 30-40 s, the rotating speed of the smashing at the low speed is 15000-20000 rpm, the single smashing time is 15-20 s, and the smashing time of the smashing at the high speed and the smashing at the low speed is 120-180 s in total.

Optionally, in the step of mashing and filtering the lung tissue mass to obtain a lung tissue mashing solution, the step of filtering includes:

filtering the smashed lung tissue by using a copper gauze with 100-120 meshes, stirring the lung tissue on the copper gauze by using a grinding rod while filtering, and collecting the filtrate.

Optionally, the step of homogenizing the lung tissue triturating solution to obtain a porcine lung tissue suspension comprises the steps of:

and (3) placing the lung tissue triturating liquid into a homogenizer, and homogenizing the lung tissue triturating liquid back and forth for three times to obtain the pig lung tissue suspension.

The preparation method of the pig lung tissue suspension provided by the invention is simple, efficient, easy to operate, time-saving and labor-saving, and can well avoid pollution and improve the cell preparation amount.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic flow chart of an embodiment of a method for preparing a suspension of porcine lung tissue according to the present invention;

fig. 2 is a schematic structural diagram of an input drainage device (fig. 2A) and a photograph of equipment (fig. 2B) when the input drainage device is used for drainage in the preparation method of the porcine lung tissue suspension provided by the invention;

FIG. 3 is a schematic view of the connection of the gastric lavage needle of FIG. 2 with the liquid outlet connector;

FIG. 4 is a photograph showing the cleaning of the outer surface of lung tissue;

FIG. 5 is a graph of lung prior to filling;

FIG. 6 is a photograph of the operation of the insertion of a lavage needle into a designated lung lobe map;

FIG. 7 is a photograph of tool equipment such as elbow and point tweezers and a grinding rod;

FIG. 8 is a picture of a lung tissue mass filling a first lung lobe;

FIG. 9 is a picture of a lung tissue mass filling the entire lung;

FIG. 10 is a photograph showing the operation of the filled lung lobe to lacerate and strip lung tissue;

FIG. 11 is a photograph of lung tissue (left) and trachea (right) before and after lung tissue is lacerated and stripped from the filled lung lobes;

FIG. 12 is a photograph of a lung tissue mass before (left) and after (right) clipping with scissors;

FIG. 13 is a photograph of a lung tissue mass prior to trituration;

FIG. 14 is a photograph of lung tissue triturating fluid filtration;

FIG. 15 is a photograph of lung tracheal tissue removed after lung tissue has been treated with scissors (left) and bent-tip forceps (right);

fig. 16 is a photograph of the connective tissue trapped by the copper gauze after lung tissue treatment with scissors (left) and elbow pointed forceps (right).

Reference numerals illustrate:

1	first container	4	Pump with a pump body
				2	Infusion tube	5	Second container
3	Stomach irrigation needle	6	Thermometer

The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The conventional preparation method of lung tissue suspension mainly adopts mechanical methods, such as shearing method and grinding method, can realize separation of cells or microorganisms, and is mostly used for laboratory researches, but the conventional preparation method mainly has the following problems that pollution exists in the preparation process, and non-target tissues such as: connective tissue and the like are easy to be mixed in cell suspension, the preparation amount is unstable, the preparation amount is small, the operation time is too long, pollution is difficult to control, and the like. In view of the above, the present invention provides a method for preparing a porcine lung tissue suspension, and fig. 1 shows an embodiment of a method for preparing a porcine lung tissue suspension according to the present invention. Referring to fig. 1, in this embodiment, the preparation method of the porcine lung tissue suspension includes the following steps:

step S10, obtaining a pig lung sample containing lung and trachea;

step S20, injecting a culture medium into a lung main air pipe of the pig lung sample to obtain pig lung filled with the culture medium;

s30, using elbow pointed forceps to scratch and strip the pig lung filled with the culture medium to obtain a lung tissue block;

step S40, mashing and filtering the lung tissue blocks to obtain lung tissue mashing liquid;

and step S50, homogenizing the lung tissue mashing liquid to obtain the pig lung tissue suspension.

The preparation method of the pig lung tissue suspension provided by the invention is simple, efficient, easy to operate, time-saving and labor-saving, and can well avoid pollution and improve the cell preparation amount.

Preferably, in some embodiments of the present invention, in the step of obtaining the pig lung sample containing lung and trachea in step S10, the main trachea is clamped by the rope, the sample is sealed, and refrigerated transportation is performed, so that pollution possibly caused in the sampling process can be effectively reduced. Specifically, step S10 includes: under the anesthesia state, the experimental pig is subjected to front cavity bleeding and death, the dissecting table is inverted, and the chest is opened along the chest collarbone; cutting off collarbone, cutting open epidermis at two sides of larynx along main trachea, and separating main trachea; tying the main airway with a wire at a distance of about 2cm from the larynx at the proximal end of the lung (see figure 5); separating heart, cutting main trachea along edge of larynx, taking out chest of pig with lung and trachea as a whole; the outer surface (shown in FIG. 4) was washed with PBS, and a sample of porcine lung containing lungs and air ducts was obtained, stored in a sealed condition, and refrigerated and returned to the laboratory for use. In other embodiments of the present invention, the step S10 of obtaining a pig lung sample containing lung and trachea may be directly providing the collected pig lung sample.

In some embodiments of the present invention, in the step S20, a power pump infusion method is adopted in the process of inputting the culture medium into the main air tube of the pig lung sample, the gastric lavage needle is used to replace the medical infusion tube needle to drain and input the culture medium into the main air tube of each lung lobe of the lung tissue, and a device capable of automatically pumping and adjusting the infusion amount is provided, so that stability and controllability of inputting the culture medium can be realized, and the time, effort and efficiency of the input process can be reduced, and the probability of causing sample pollution can be reduced. Specifically, in step S20, the culture medium is injected into the main lung trachea of the pig lung sample by using an input drainage device, as shown in fig. 2A and 2B, the input drainage device includes an input system and a condensation system, the input system includes a first container 1 (specifically may be an infusion bottle), an infusion tube 2 and a pump 4, the first container 1 is used for containing the culture medium, one end of the infusion tube 2 is communicated into the first container 1, the other end of the infusion tube 2 is provided with a gastric perfusion needle 3 for inserting into the pig lung sample (see fig. 6), the intravenous infusion needle portion of the disposable infusion set is replaced by a gastric perfusion needle commonly used in animal experiments, the needle plug of the gastric perfusion needle 3 is connected to the liquid outlet connector (see fig. 3) of the infusion tube 2, and the culture medium can be directionally drained into the bronchi of each lobe of the lung tissue by using the gastric perfusion needle 3. The middle part of the infusion tube 2 is connected in a power clamping groove of the pump 4 so as to pump the culture medium in the first container 1 through the pump 4 and convey the culture medium into the pig lung sample, and a controller is arranged on the pump 4 and used for starting or stopping infusion and controlling the infusion flow rate; the condensing system comprises a second container 5 and a thermometer 6, wherein the second container 5 is used for containing an ice-water mixture and the first container 1, so that the first container 1 is in an ice-water mixture environment, and the thermometer 6 is arranged in the second container 5 and used for detecting the temperature in the second container 5.

The drainage input device provided by the invention comprises an input system and a condensing system, wherein the condensing system is arranged to ensure that a condensing culture medium is input into the pig lung sample so as to ensure the quality of pig lung tissue suspension prepared in the later period; the controller is arranged on the pump 4, so that the start or stop of the injection of the culture medium into the pig lung sample can be controlled, the injection speed and the injection quantity of the culture medium are controlled, the whole input process of the culture medium is more controllable, and the method is convenient and efficient.

Specifically, in some embodiments of the present invention, the step of injecting the culture medium into the pig lung sample using the drainage input device comprises: the temperature in the second container 5 is monitored in real time by using the thermometer 6, and the temperature of the condensing system is kept at 2-8 ℃ so as to ensure that the culture medium injected into the pig lung sample is in a low-temperature state, and further ensure the activity of microorganisms in the prepared pig lung tissue suspension. Then, in a controllable environment, the first container 1, the infusion tube 2, the gastric lavage needle 3 and the pump 4 are assembled to obtain the input system, air in the infusion tube 2 is exhausted, a start-stop switch on the pump 34 is stopped, the first container 1 is filled with the culture medium, and then the culture medium is placed in the second container 5, so that the drainage input system is assembled. The pump 4 is provided with a knob and a start-stop key to control the speed of injecting the culture medium into the pig lung sample and start-stop, and the filling degree of the lung tissue is regulated together with the gastric lavage needle (see fig. 5 and 9), or the preparation sequence of the target tissue in each lung lobe of the lung tissue is designated (see fig. 8).

The culture medium is filled in the lung tissue, so that the lung contraction space is small, the elasticity is weak, the follow-up efficient drawing and separating of the lung tissue are facilitated, the filling of the culture medium is more favorable for coexistence of microorganisms which want to be obtained in the lung tissue, and the preparation quantity is improved. It is understood that the variety of the culture medium can be different according to the application difference of the pig lung tissue suspension, and antibiotics with proper concentration can be added, so that pollution is avoided, and other related reagents are required to be sterile.

Further, in some embodiments of the present invention, the initial flow rate of the medium injected into the lung main air duct of the pig lung sample is 150-250 mL/min, until the lung filling degree reaches 80-90%, the infusion flow rate is adjusted to 15-25 mL/min until the lung is completely filled (fig. 5 shows a lung diagram before filling, fig. 8 shows a lung tissue mass filling picture of the first lung lobe, fig. 9 shows a lung tissue mass filling picture of the whole lung), the injection of the medium is stopped, the needle is taken out, and the lung main air duct is clamped. The arrangement is beneficial to rapid and efficient filling of the lung, avoids outflow of culture medium, and ensures the preparation quantity and quality of the tissue suspension. The rapid filling is beneficial to shortening the preparation time and improving the preparation efficiency, the prepared tissue suspension has better activity, and if the flow rate is too small, the rapid injection of the culture medium into the lung is not beneficial, the preparation process takes a long time, and the quality is reduced; when the lung filling degree is 80-90%, the flow rate is adjusted to 15-25 ml/min, so that the input amount of the culture medium is controlled, the preparation amount of the target tissue suspension is better, and when the flow rate is too high, the input amount of the culture medium is not controlled in time, the outflow of the culture medium is caused, and the preparation amount of the tissue suspension is reduced.

The input drainage device uses the gastric lavage needle to replace the medical infusion set needle, is used for directionally draining the culture medium into the bronchi of each lung lobe of the lung tissue, is preferably a 16-20-number elbow gastric lavage needle, and is more beneficial to drainage.

In some embodiments of the invention, the amount of medium injected per 100g of the porcine lung sample is 500-1500 mL, preferably 1000mL of medium injected per 100g of the porcine lung sample.

In some embodiments of the present invention, a large number of experiments prove (shown in fig. 12, 15 and 16) that the elbow pointed forceps are used to replace the conventional scissors to separate the lung tissue from the trachea, the obtained tissue block is small and uniform, the lung tissue attached to the larger lung trachea is peeled cleanly, almost all the lung tissue can be peeled, the preparation amount of the lung tissue suspension is improved, in addition, the use of the elbow pointed forceps for peeling the lung tissue can greatly reduce the introduction of connective tissues such as the trachea, the subsequent mashing time is reduced, the quality of the prepared lung tissue suspension is ensured, the filtration efficiency of mashing the lung tissue is improved, the operation resistance in the homogenization process is greatly reduced, the whole subsequent process is time-saving, labor-saving and efficient, and meanwhile, the safety of the pointed forceps is better.

Specifically, step S30 includes: placing the pig lung filled with the culture medium in a shallow mouth container, puncturing the lung tissue along the parallel direction of the main air pipe of the lung by using an elbow of an elbow tip forceps (shown in figure 7), repeating the operations of puncturing and peeling the lung, so that each lung lobe tissue is scratched into independent small blocks of lung tissue separated from the air pipe, and collecting the lung lobe tissue together with the culture medium injected into the lung tissue in the shallow mouth container (shown in figures 10 and 13); at the same time, a small amount of the lung tissue pieces attached to the trachea are sequentially peeled off by continuing to clamp the bent parts using the sharp forceps, so that almost all the lung tissue pieces are obtained (shown in fig. 11 and 13).

In some embodiments of the present invention, the lung tissue is circularly mashed in step S40, preferably using a stainless steel masher, with a low-speed mashing and then a high-speed mashing, wherein the high-speed mashing is performed at a rotational speed of 20000 to 25000rpm, a single mashing time of 30 to 40S, the low-speed mashing is performed at a rotational speed of 15000 to 20000rpm, a single mashing time of 15 to 20S, and the mashing times of the high-speed mashing and the low-speed mashing are in total 120 to 180S. Namely, the low-speed mashing is firstly carried out, the high-speed mashing is carried out after the low-speed mashing is finished, the low-speed mashing is carried out after the high-speed mashing, the circulation is carried out for 2 to 3 times, and the total mashing time is 120 to 180 seconds. Therefore, the method of alternately carrying out high-speed trituration and low-speed trituration is beneficial to avoiding the destruction of local high temperature to microorganisms in the prepared cell suspension caused by long-time high-speed trituration so as to ensure the quality of the prepared lung tissue suspension.

In some embodiments of the present invention, the filtering in step S40 is specifically: the crushed lung tissue was filtered through a copper gauze of 100 to 120 mesh, and the filter was collected by stirring the filter on the copper gauze with a grinding rod (shown in FIG. 7). In this way, the stirring effect of the grinding rod not only can dredge the blocked copper yarn meshes, but also can collect connective tissues causing blocking holes, so that the filtering effect of the copper yarn net is improved, and the use times of the copper yarn net are increased.

In some embodiments of the present invention, the homogenizing in step S50 is specifically: and (3) placing the lung tissue triturating liquid into a homogenizer, and homogenizing the lung tissue triturating liquid back and forth for three times to obtain the pig lung tissue suspension.

By filtering the copper gauze and homogenizing the copper gauze by a homogenizer, non-target tissues introduced in the preparation process are gradually removed, and a tissue suspension with better purity is obtained.

In summary, the invention controls the input of the culture medium (shown in fig. 2 and 3) by adopting a power pump infusion mode, controls the flow direction of the culture medium (shown in fig. 6) by using the gastric lavage needle 3 to fill lung tissues, uses the elbow forceps to scratch and strip lung tissue blocks (shown in fig. 10, 11 and 13), smashes the collected lung tissue blocks, and finally realizes the standardized operation procedure of alveolar tissue suspension preparation by filtering (shown in fig. 14) and homogenizing operation, thereby achieving the purposes of simplicity, high efficiency, easy operation, time and labor saving, pollution avoidance and preparation quantity improvement. It should be noted that, the objects such as elbow pointed forceps and gastric lavage needle used in the technical scheme of the invention all need to be sterilized materials or sterile objects, and the preparation process of the lung tissue suspension should be carried out in a controllable environment, such as an ultra-clean workbench, a biosafety cabinet and the like.

The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.

Example 1

(1) The method comprises the following steps of separating and obtaining a pig lung sample containing lung and air ducts from a pig body:

under the anesthesia state of the experimental pig, the front cavity is exsanguinated and is fatal, the dissecting table is inverted, and the chest is opened along the chest collarbone; cutting off collarbone, cutting open epidermis at two sides of larynx along main trachea, and separating main trachea; tying the main airway (as shown in figure 5) about 2cm from the larynx at the proximal end of the lung; separating heart, cutting main trachea along edge of larynx, taking out chest of pig with lung and trachea as a whole; the outer surface was washed with PBS, sealed and refrigerated and taken back to the laboratory.

(2) Injecting a culture medium into a pig lung sample by using an input drainage device, wherein the steps are as follows:

taking out pig lungs, placing the pig lungs in an ultra-clean bench, washing the outer surface with PBS, weighing, placing the volume of an input culture medium (controlling the lung sample of each 100g to be correspondingly injected with 1000mL of culture medium) in a fluid infusion bottle, taking a disposable sterile infusion tube to be connected with an output tube of the fluid infusion bottle, placing the fluid infusion bottle filled with the culture medium at the right center of an ice-water mixture, bypassing a peristaltic pump, connecting a gastric lavage needle at the other end, starting an operation switch of the pump, evacuating air in the infusion tube, and suspending the operation switch; then the stomach filling needle head stretches into the main air pipe, the infusion pipe is connected, the flow rate of the pump is set to be 200mL/min, the running switch on the pump is turned on, PBS is injected, when the filling degree of the whole lung is 85%, the flow rate is adjusted to be 20mL/min, the running switch on the pump is stopped, the stomach filling needle head is taken out, the main air pipe of the lung is clamped, and the lung filled with the culture medium is obtained.

(3) The lung tissue is scraped and stripped as follows:

placing the lung filled with the culture medium in a shallow mouth container, pricking the elbow of the elbow pointed forceps into lung tissue, scratching the lung tissue along the parallel direction of the main air pipe of the lung, repeatedly scratching the lung, scratching each lung lobe tissue into independent small lung tissue, separating from the air pipe, collecting the lung tissue together with the culture medium injected into the lung tissue, and collecting the lung tissue in the shallow mouth container (shown in figure 10); in addition, a small amount of lung tissue blocks attached to the trachea are continuously clamped and peeled one by the elbow parts of the tip forceps, so that almost all the lung tissue blocks can be obtained;

(4) The lung tissue is mashed as follows:

the method comprises the steps of mashing lung tissue blocks by using a stainless steel masher, wherein the mashing is carried out at a low speed and then at a high speed, and the mashing is carried out in a mode of alternately carrying out the mashing and the mashing in sequence, wherein the rotating speed of the mashing at the high speed is 22000rpm, the single mashing time is 40s, the rotating speed of the mashing at the low speed is 18000rpm, the single mashing time is 20s, and the mashing time of the mashing at the high speed and the mashing at the low speed is 2min in total, so that the mashed lung tissue is obtained.

(5) The lung tissue is mashed and filtered as follows:

after the smashed lung tissue is obtained, the smashed lung tissue is filtered by a 100-mesh copper gauze, and the smashed lung tissue is obtained by stirring and accelerating the filtering on the copper gauze while filtering.

(6) The homogenization treatment of the lung tissue triturating fluid comprises the following steps:

the obtained lung tissue triturating liquid is homogenized by a homogenizer for three times, thus obtaining lung tissue suspension.

Example 2

The lung tissue is positive with the blue ear virus selected from the laboratory, and the other exogenous viruses are negative lung tissue.

Comparative example 1

The procedure was the same as in example 1, except that scissors were used instead of bent-tipped forceps in step (3) (shown in fig. 12, 15 and 16).

Comparative example 2

The procedure was the same as in example 1, except that step (2) was omitted and the obtained porcine lung tissue sample was directly stripped in step (3).

Comparative example 3

The procedure is the same as in example 1, except that in step (4) the lung tissue is mashed at 22000rpm for 2min continuously.

Comparative example 4

The lung tissue is positive to the blue ear virus in the laboratory, other exogenous viruses are negative lung tissue, 1g of lung tissue is weighed, sheared by scissors, the tissue is ground in a mortar by a grinding rod matched with quartz sand, and the lung tissue is collected and filtered to obtain lung tissue suspension.

The volume of lung tissue suspension harvested in example 1 and comparative examples 1-3, the time taken for the whole preparation process, and the temperature of the lung tissue trituration solution at the end of trituration were counted, and the results are shown in Table 1.

Table 1 relevant test data for the preparation in example 1 and comparative examples 1-3

The effects of the prepared lung tissue suspensions were determined by performing virus isolation and virus content detection on the samples prepared in example 2 and comparative example 4, and the results of the toxicity measurement of the lung tissue suspensions prepared in example 2 and comparative example 4, the volumes of the lung tissue suspensions obtained in example 2 and comparative example 4, the time taken for the whole preparation process, the temperature of the lung tissue trituration liquid at the end of trituration, and the toxicity detection results of the lung tissue suspensions were counted, and the results are shown in table 2.

Table 2 relevant test data for the preparation of example 2 and comparative example 4

As can be seen from the test data in table 1: in the embodiment 1, the preparation amount of the pig lung tissue suspension can reach 1832ml, the mashing temperature is 15 ℃, the whole preparation process takes 1.6 hours, and the prepared pig lung tissue suspension is uniform and controllable.

In comparative example 1, scissors are used for replacing elbow pointed forceps, the preparation amount of the pig lung tissue suspension can reach 1408ml, the mashing temperature is 31 ℃ and is higher than the room temperature by 25 ℃, the whole preparation process takes 3.8 hours, and the prepared pig lung tissue suspension is uniform. The whole operation process mainly has incomplete removal of the tracheal tissue, nonuniform size of the lung tissue block, long mashing time, large interception amount of copper gauze filtration, blockage, slow filtration and long time consumption of filtration; homogenization is laborious, time-consuming, and hurts hands.

In comparative example 2, the lung tissue is not filled with culture medium, the lung tissue is directly scratched and peeled by using a pair of pointed forceps, the preparation amount of the pig lung tissue suspension can reach 1580ml, the mashing temperature is 35 ℃ and is higher than the room temperature by 25 ℃, the whole preparation process takes 2.4 hours, and the prepared pig lung tissue suspension is uniform. The whole lung tissue block has long scratching and stripping time, and the healthy lung has large elasticity, so that the scratching is not facilitated; a small amount of lung tissue attached to the trachea is not completely peeled off, and the like.

In comparative example 3, the preparation amount of the pig lung tissue suspension can reach 1740ml without intermission during trituration, the trituration temperature is 21 ℃ and slightly lower than the room temperature of 25 ℃, the whole preparation process takes 1.5 hours, and the prepared pig lung tissue suspension is nonuniform. The prepared pig lung tissue suspension is uniform after long-time high-speed crushing, but the local instantaneous temperature exceeds 21 ℃ in the whole operation process, and the later use of the pig lung tissue suspension can be influenced.

As can be seen from the test data in table 2: in example 2 and comparative example 4, the same lung, lung tissue suspensions obtained in different ways, had little difference in the amount of pig lung tissue suspension prepared; the temperature of the traditional grinding method is closer to the room temperature during preparation and lower than the temperature during mashing by the method, but the method has little influence on microorganisms in the preparation suspension; the difference of the virus content measured by using Marc-145 cells for 5 passages of the obtained lung tissue suspension is not obvious. Only in the whole preparation process, the traditional grinding time is more than 4.5 hours, the grinding operation is extremely laborious, and a plurality of cooperations are difficult to finish the grinding and separation of the whole lung tissue, so that the traditional grinding method is applicable to the preparation of a very small amount of lung tissue samples, and the defects of difficulty in realizing amplified preparation and the like are overcome;

the preparation method provided by the invention is used for preparing the pig lung tissue suspension, the preparation amount of the pig lung tissue suspension can reach 1832ml, the temperature is 12-20 ℃ during mashing, and the preparation time is 1.6-1.8 h; the preparation amount of the lung tissue suspension can basically ensure uniformity and can be used later.

Application example 1

The method of the embodiment 1 of the invention is adopted to count corresponding data of a batch of healthy piglets with weight of about 15kg for preparing the pig lung tissue suspension, and the data of the piglets with weight of 12.5kg for preparing the pig lung tissue suspension are listed, and the conclusion is as follows:

1. multiple researches show that the volume of the lung tissue suspension for preparing the piglet with the weight of about 15kg through the operation procedure provided by the invention can reach 1200-2000 ml, and the preparation time is 1.3-2.2 h. The preparation process in the laboratory can be controlled to be 2.0 hours, and the prepared suspension is pollution-free and can be used for subsequent experimental study.

2. A healthy piglet with a weight of 12.0kg was selected, anesthetized, bled and fatigued, and the lung was taken, weighed at 154.2g, refrigerated and transported back to the laboratory for pig lung tissue suspension preparation. Cleaning and disinfecting the surface of the lung, filling the lung tissue, scraping and peeling the lung tissue blocks by using elbow sharp forceps, smashing, filtering and homogenizing to prepare pulmonary alveolus tissue suspension, wherein the total time of the experimental process is about 1.9h, the total amount of the prepared pig lung tissue suspension is 1608ml, and subsequent researches show that the lung tissue suspension can be used.

The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. A method for preparing a suspension of porcine lung tissue, comprising the steps of:

obtaining a pig lung sample containing lung and trachea;

injecting a culture medium into a lung main air pipe of the pig lung sample to obtain pig lung filled with the culture medium;

using elbow pointed forceps to scratch and strip the pig lung filled with the culture medium to obtain a lung tissue block;

mashing and filtering the lung tissue blocks to obtain lung tissue mashing liquid;

homogenizing the lung tissue triturating liquid to obtain the pig lung tissue suspension.

2. The method of claim 1, wherein the step of obtaining a sample of porcine lung comprising lung and trachea comprises: the main air pipe is clamped by a rope, and the sample is sealed and refrigerated for transportation.

3. The method of claim 1, wherein in the step of injecting a culture medium into the lung main trachea of the pig lung sample, an input drainage device is used to inject the culture medium into the lung main trachea of the pig lung sample, the input drainage device comprising:

the input system comprises a first container, a perfusion tube and a pump, wherein the first container is used for containing the culture medium, one end of the perfusion tube is communicated into the first container, the other end of the perfusion tube is provided with a stomach-filling needle head which is used for being inserted into the pig lung sample, the perfusion tube is connected to a power clamping groove of the pump so as to pump the culture medium in the first container through the pump and convey the culture medium into the pig lung sample, and the pump is provided with a controller which is used for starting or stopping transfusion and controlling the flow rate of the transfusion; the method comprises the steps of,

the condensing system comprises a second container and a thermometer, wherein the second container is used for containing an ice-water mixture and the first container, so that the first container is in an ice-water mixture environment, and the thermometer is arranged in the second container and used for detecting the temperature in the second container.

4. A method of preparing a porcine lung tissue suspension according to claim 3, wherein the step of injecting the culture medium into the lung main trachea of the porcine lung sample using an input drainage device:

and (3) injecting culture medium into the lung main air pipe of the pig lung sample at an initial flow rate of 150-250 mL/min until the lung filling degree reaches 80-90%, regulating the infusion flow rate to 15-25 mL/min until the lung is completely filled, stopping injecting the culture medium, taking out the needle head and clamping the lung main air pipe.

5. A method of preparing a porcine lung tissue suspension according to claim 3 wherein the lavage needle is a 16-20 gauge elbow lavage needle.

6. The method of claim 1, wherein the step of injecting a culture medium into the main lung airways of the porcine lung sample to obtain a culture medium-filled porcine lung comprises:

the amount of the culture medium injected into each 100g of the pig lung sample is 500-1500 mL.

7. The method of preparing a porcine lung tissue suspension according to claim 1, wherein the step of lacerating and dissecting the porcine lung of the filled medium using bent tip forceps to obtain a lung tissue mass comprises:

placing the pig lung filled with the culture medium in a shallow mouth container, pricking lung tissues with the elbow of the elbow pointed forceps, tearing the lung tissues along the parallel direction of the main air pipe of the lung, repeating the operation of tearing and stripping the lung tissues, so that each lung lobe tissue is torn into independent small lung tissues separated from the air pipe, and simultaneously, stripping the lung tissue blocks attached to the air pipe one by using the elbow parts of the pointed forceps, thereby obtaining the lung tissue blocks.

8. The method of claim 1, wherein the step of mashing the lung tissue pieces in the step of mashing and filtering the lung tissue pieces to obtain a lung tissue mashing solution comprises:

the lung tissue is circularly smashed by using a stainless steel smashing machine in a mode of smashing at a low speed and smashing at a high speed, wherein the rotating speed of the smashing at the high speed is 20000-25000 rpm, the single smashing time is 30-40 s, the rotating speed of the smashing at the low speed is 15000-20000 rpm, the single smashing time is 15-20 s, and the smashing time of the smashing at the high speed and the smashing at the low speed is 120-180 s in total.

9. The method of claim 1, wherein in the step of mashing and filtering the pieces of lung tissue to obtain a lung tissue mashing solution, the step of filtering comprises:

filtering the smashed lung tissue by using a copper gauze with 100-120 meshes, stirring the lung tissue on the copper gauze by using a grinding rod while filtering, and collecting the filtrate.

10. The method of preparing a porcine lung tissue suspension according to claim 1, wherein the step of homogenizing the lung tissue trituration solution to obtain a porcine lung tissue suspension comprises:

and (3) placing the lung tissue triturating liquid into a homogenizer, and homogenizing the lung tissue triturating liquid back and forth for three times to obtain the pig lung tissue suspension.

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