阅读说明：本技术 一种规模化制备鸡胚成纤维细胞的方法 (Method for preparing chicken embryo fibroblasts in large scale ) 是由 崔松奇 魏文江 田春利 姚沾沾 余海涛 张丹丹 李娥 张娜 李晓静 于 2021-09-09 设计创作，主要内容包括：本发明涉及一种规模化制备鸡胚成纤维细胞的方法。包括以下步骤：(1)挑选鸡胚并进行初步消毒；(2)对鸡胚进行再次消毒；(3)取鸡胚；(4)处理鸡胚；(5)清洗鸡胚；(6)消化鸡胚；(7)组织细胞离心；(8)组织细胞重悬；(9)组织细胞过滤；(10)分装；(11)细胞培养。本发明提供了一种规模化制备鸡胚成纤维细胞的方法,与现有的制备方法相比,本制备方法实现了对鸡胚成纤维细胞的无菌制备、提升规模效率制备以及降低成本制备,实现了低制备成本下的高效、规模化制备,保证了鸡胚成纤维细胞的质量,解决了生物制品规模化生产的瓶颈问题,满足工业化生产对鸡胚成纤维细胞的需要。(The invention relates to a method for preparing chicken embryo fibroblasts in a large scale. The method comprises the following steps: (1) selecting chicken embryos and carrying out primary disinfection; (2) sterilizing the chick embryo again; (3) taking a chicken embryo; (4) treating the chick embryos; (5) cleaning chicken embryos; (6) digesting the chick embryo; (7) centrifuging tissue cells; (8) resuspending the tissue cells; (9) filtering the tissue cells; (10) subpackaging; (11) and (5) culturing the cells. Compared with the existing preparation method, the preparation method realizes aseptic preparation of the chicken embryo fibroblasts, improves scale efficiency preparation and reduces cost preparation, realizes efficient and scale preparation at low preparation cost, ensures the quality of the chicken embryo fibroblasts, solves the bottleneck problem of scale production of biological products, and meets the requirement of industrial production on the chicken embryo fibroblasts.)

1. A method for preparing chicken embryo fibroblasts in a large scale is characterized by comprising the following steps: comprises the following steps of (a) carrying out,

(1) selecting chicken embryos and carrying out primary disinfection: selecting a plurality of SPF (specific pathogen free) chick embryos which are well developed in a chick embryo pre-incubation chamber according to embryos, sending the chick embryos into a disinfection chamber, disinfecting the surfaces of the chick embryos by using disinfectant spray, and then transferring the chick embryos into a greenhouse;

(2) and (3) disinfecting the chick embryos again: taking out the chick embryo from the greenhouse, dipping iodine tincture by using a brush, disinfecting the upper part of the chick embryo, and deiodinating the chick embryo by using absolute ethyl alcohol after the iodine tincture is dried;

(3) taking chicken embryos: opening the air chamber of the chick embryo, taking out the chick embryo, and putting the chick embryo into a plate;

(4) treating chicken embryos: clamping and removing the eyes of the chick embryo by using a tool;

(5) cleaning chicken embryos: adding the chicken embryos into a cleaner, injecting the Hankel solution into the cleaner, shaking the chicken embryo cleaner to fully mix the chicken embryos with the Hankel solution, then sucking out the Hankel solution in the cleaner, and repeating the process of cleaning the chicken embryos for multiple times;

(6) digesting chicken embryos: pouring the cleaned chick embryos into a magnetic digestion bottle, stirring at a high rotating speed to completely break the chick embryos in the digestion bottle into pasty suspended tissues, then adjusting to a low rotating speed, stirring, adding pancreatin, and fully digesting the chick embryo tissues under the action of the pancreatin;

(7) tissue cell centrifugation: digesting the chicken embryo suspension tissue, adding serum into the digestive juice under the aseptic condition to neutralize pancreatin, and centrifuging by adopting a centrifuge;

(8) resuspending the tissue cells: discarding the centrifuged supernatant, adding the cell growth liquid into the centrifuge tube, shaking the centrifuge tube to separate the histiocytes from the tube wall of the centrifuge tube, adding the histiocyte into the mixing bottle, and shaking the mixing bottle to make the histiocyte become suspension;

(9) filtering tissue cells: sucking the tissue cell resuspension into a subpackaging tank with a gauze bag type filtering device, filtering and intercepting undigested chick embryo tissues in the cell suspension by the cell suspension through the gauze bag type filtering device, and then supplementing a cell culture solution into the subpackaging tank;

(10) subpackaging: subpackaging the cell suspension in the subpackaging tank to a plurality of cell factories;

(11) cell culture: and (4) placing the cell factory into a greenhouse for static culture to obtain the monolayer chicken embryo fibroblast.

2. The method for large-scale preparation of chicken embryo fibroblasts as claimed in claim 1, which is characterized in that: in the step (1), the selected SPF chick embryos are 9 days old, and the room temperature in a greenhouse is 38 ℃.

3. The method for large-scale preparation of chicken embryo fibroblasts as claimed in claim 2, which is characterized in that: and (2) taking out the chick embryos from the greenhouse for the next day after the chick embryos are placed in the greenhouse, and disinfecting the middle upper parts of the chick embryos by iodine.

4. The method for large-scale preparation of chicken embryo fibroblasts as claimed in claim 3, which is characterized in that: in the step (5), a plurality of obtained chick embryos are subpackaged into a plurality of cleaners, a liquid inlet on a three-way cover at the top of each cleaner is connected with a cleaning liquid storage tank by adopting a multi-connection pipeline, an air suction port on the three-way cover at the top of each cleaner is connected by adopting the multi-connection pipeline, a vacuum pump is arranged on the multi-connection pipeline, a liquid outlet on the three-way cover at the top of each cleaner is connected with a waste liquid storage tank by adopting the multi-connection pipeline, and a liquid suction pump is arranged on the multi-connection pipeline; the number of washes was three.

5. The method for large-scale preparation of chicken embryo fibroblasts as claimed in claim 4, which is characterized in that: in the step (6), high-speed stirring and low-speed stirring are carried out at the temperature of 38 ℃, the duration time of the high-speed stirring is 5min, and the duration time of the low-speed stirring is 15 min; after stirring at a low rotating speed, removing a melting bottle to observe the sediment at the bottom, and taking the cerebro-sphere tissue, the liver, the vertebra bone, the leg part and the wing part bone in the sediment at the bottom which are clearly visible and no muscle tissue on the bone as the end point of the step of judging the digestion of the chick embryo.

6. The method for large-scale preparation of chicken embryo fibroblasts as claimed in claim 5, which is characterized in that: in the step (7), the weight of the added serum is 3% of the total weight of the digestive juice; the rotation speed of the centrifuge is 3000r/min, and the centrifugation duration is 10 min.

7. The method for large-scale preparation of chicken embryo fibroblasts as claimed in claim 6, which is characterized in that: in the step (9), the volume of the subpackaging tank is 50L, and the histiocyte resuspension liquid of every 500 chick embryos is injected into one subpackaging tank; the gauze bag type filtering device in the subpackaging tank comprises 18 layers of gauzes, and cell culture solution is supplemented to 50L scale marks of the subpackaging tank after injection and filtration of the histiocyte resuspension of the chick embryos are completed.

8. The method for large-scale preparation of chicken embryo fibroblasts as claimed in claim 7, wherein: in the step (10), the outlet of each sub-packaging tank is connected with a liquid distribution pipe with a plurality of liquid distribution heads, each liquid distribution head is connected to the injection port of one cell factory, and the cell suspension liquid of a plurality of cell factories is sub-packaged at one time.

9. The method for large-scale preparation of chicken embryo fibroblasts as claimed in claim 8, wherein: in the step (11), the temperature of a culture greenhouse of the cell factory is 38 ℃, and the culture time is 18-24 h.

Technical Field

The invention belongs to the technical field of biological products for livestock, and particularly relates to a method for preparing chicken embryo fibroblasts in a large scale.

Background

The chicken embryo fibroblast is a common primary cell for biological product production and inspection, and the current culture process of the chicken embryo fibroblast has various modes: there are culture in square flasks for laboratory experiments, spinner flask culture for vaccine production, cell factory culture, and bioreactor culture, but the preparation of primary fibroblasts is an insurmountable step regardless of the culture method. The square flask culture refers to cell culture in a square flask, and the square flask (T-flash) is a special bottle used in animal cell culture. When in use, the bottle is horizontally placed, the upper plane and the lower plane are parallel, the side surface is provided with a threaded opening, and cells can adhere to the inner lower surface of the bottle after being inoculated. The spinner flask culture is to adopt a rotary spinner flask to culture cells, the spinner flask is fixed on a bracket, the inclination is 5-10 degrees, or the spinner flask is placed between a row of rotating shafts, so that the spinner flask slowly rotates along with the rotating shafts at 6-12 turns per hour. The cells are attached to the periphery of the glass wall, the cells adhere to the wall and grow on the surface with only one surface area, and the culture solution flows through rotation, so that the cells can absorb nutrition and metabolize, and the cells can contact gas organically, so that the cells can breathe and generate redox, and the cells can grow and reproduce. Cell factory culture refers to the culturing of cells in a cell factory. Bioreactor culture refers to culturing in a dedicated bioreactor.

As mentioned above, the conventional methods for preparing chicken embryo fibroblasts are more suitable for laboratory-level refinement operations, because the batch size of the chicken embryo fibroblasts is small and the process is easy to control under laboratory conditions. However, for the mass production with strict requirements on time and efficiency, strict requirements on production cost and requirements on preparation scale, the process for preparing the chick embryo fibroblasts in small batches is not suitable, and an alternative solution capable of realizing mass preparation is needed.

The scale preparation of chicken embryo fibroblasts needs to solve three problems: sterile preparation, improved scale efficiency and reduced preparation cost. Firstly, aseptic preparation is the most important in the production of industrial biological products, and dozens or even millions of yuan RMB are put into various raw and auxiliary materials at one time, so that the pollution probability is reduced to the minimum; secondly, compared with the method that dozens of chick embryos are operated at one time in a laboratory, the treatment capacity of thousands of chick embryos needs to be large in large-scale production, and the preparation work of cells needs to be completed within four hours and five hours, so that the process needs to be continuously optimized, and the preparation efficiency is improved; moreover, enterprises aim to be profitable, and for the production link, the labor input needs to be reduced, the yield of chick embryo cells in a unit is improved, and the production cost is reduced, so that the profit of products is maximized. The existing preparation process is difficult to meet the requirements, so that technological innovation and technological optimization are continuously carried out on the basis of the traditional chicken embryo fibroblast preparation method to solve the bottleneck of large-scale production of biological products and further meet the requirement of industrial production on the chicken embryo fibroblasts.

Disclosure of Invention

The invention provides a method for preparing chicken embryo fibroblasts in a large scale for solving the technical problems in the prior art, realizes large-scale, aseptic and low-cost preparation of the chicken embryo fibroblasts, and meets the requirements of industrial production on the chicken embryo fibroblasts.

The technical scheme adopted by the invention for solving the technical problems in the prior art is as follows: a method for preparing chicken embryo fibroblasts in a large scale comprises the following steps of (1) selecting chicken embryos and carrying out primary disinfection: selecting a plurality of SPF (specific pathogen free) chick embryos which are well developed in a chick embryo pre-incubation chamber according to embryos, sending the chick embryos into a disinfection chamber, disinfecting the surfaces of the chick embryos by using disinfectant spray, and then transferring the chick embryos into a greenhouse; (2) and (3) disinfecting the chick embryos again: taking out the chick embryo from the greenhouse, dipping iodine tincture by using a brush, disinfecting the upper part of the chick embryo, and deiodinating the chick embryo by using absolute ethyl alcohol after the iodine tincture is dried; (3) taking chicken embryos: opening the air chamber of the chick embryo, taking out the chick embryo, and putting the chick embryo into a plate; (4) treating chicken embryos: clamping and removing the eyes of the chick embryo by using a tool; (5) cleaning chicken embryos: adding the chicken embryos into a cleaner, injecting the Hankel solution into the cleaner, shaking the chicken embryo cleaner to fully mix the chicken embryos with the Hankel solution, then sucking out the Hankel solution in the cleaner, and repeating the process of cleaning the chicken embryos for multiple times; (6) digesting chicken embryos: pouring the cleaned chick embryos into a magnetic digestion bottle, stirring at a high rotating speed to completely break the chick embryos in the digestion bottle into pasty suspended tissues, then adjusting to a low rotating speed, stirring, adding pancreatin, and fully digesting the chick embryo tissues under the action of the pancreatin; (7) tissue cell centrifugation: digesting the chicken embryo suspension tissue, adding serum into the digestive juice under the aseptic condition to neutralize pancreatin, and centrifuging by adopting a centrifuge; (8) resuspending the tissue cells: discarding the centrifuged supernatant, adding the cell growth liquid into the centrifuge tube, shaking the centrifuge tube to separate the histiocytes from the tube wall of the centrifuge tube, adding the histiocyte into the mixing bottle, and shaking the mixing bottle to make the histiocyte become suspension; (9) filtering tissue cells: sucking the tissue cell resuspension into a subpackaging tank with a gauze bag type filtering device, filtering and intercepting undigested chick embryo tissues in the cell suspension by the cell suspension through the gauze bag type filtering device, and then supplementing a cell culture solution into the subpackaging tank; (10) subpackaging: subpackaging the cell suspension in the subpackaging tank to a plurality of cell factories; (11) cell culture: and (4) placing the cell factory into a greenhouse for static culture to obtain the monolayer chicken embryo fibroblast.

The invention has the advantages and positive effects that:

compared with the existing preparation method, the invention provides the method for aseptic preparation of the chicken embryo fibroblasts, preparation with improved scale efficiency and preparation with reduced cost. Through the step of setting up the chicken embryo disinfection, realized disinfecting and surface drying to the chicken embryo and handled, effectively prevent that the surface from remaining liquid from getting into in the air chamber when the air chamber of sequent chicken embryo is opened, reduced the risk that the chicken embryo received the pollution. By optimizing the chicken embryo processing mode, the time required by chicken embryo processing is saved, the number of the available chicken embryo cells which can be obtained is increased, the yield of the chicken embryo fibroblasts is increased, the optimized chicken embryo processing mode can also reduce the time of exposing the chicken embryos to the external environment, and the pollution risk is further reduced. The method effectively realizes the acquisition of effective chick embryo cells by the steps of cleaning chick embryos, digesting the chick embryos, centrifuging histiocytes, resuspending the histiocytes, filtering the histiocytes and the like. The preparation method is a large-scale preparation method, and compared with the existing laboratory-level preparation method, the method realizes efficient and large-scale preparation at low preparation cost, ensures the quality of the chick embryo fibroblasts, solves the bottleneck problem of large-scale production of biological products, and meets the requirements of industrial production on the chick embryo fibroblasts.

Preferably: in the step (1), the selected SPF chick embryos are 9 days old, and the room temperature in a greenhouse is 38 ℃.

Preferably: and (2) taking out the chick embryos from the greenhouse for the next day after the chick embryos are placed in the greenhouse, and disinfecting the middle upper parts of the chick embryos by iodine.

Preferably: in the step (5), a plurality of obtained chick embryos are subpackaged into a plurality of cleaners, a liquid inlet on a three-way cover at the top of each cleaner is connected with a cleaning liquid storage tank by adopting a multi-connection pipeline, an air suction port on the three-way cover at the top of each cleaner is connected by adopting the multi-connection pipeline, a vacuum pump is arranged on the multi-connection pipeline, a liquid outlet on the three-way cover at the top of each cleaner is connected with a waste liquid storage tank by adopting the multi-connection pipeline, and a liquid suction pump is arranged on the multi-connection pipeline; the number of washes was three.

Preferably: in the step (6), high-speed stirring and low-speed stirring are carried out at the temperature of 38 ℃, the duration time of the high-speed stirring is 5min, and the duration time of the low-speed stirring is 15 min; after stirring at a low rotating speed, removing a melting bottle to observe the sediment at the bottom, and taking the cerebro-sphere tissue, the liver, the vertebra bone, the leg part and the wing part bone in the sediment at the bottom which are clearly visible and no muscle tissue on the bone as the end point of the step of judging the digestion of the chick embryo.

Preferably: in the step (7), the weight of the added serum is 3% of the total weight of the digestive juice; the rotation speed of the centrifuge is 3000r/min, and the centrifugation duration is 10 min.

Preferably: in the step (9), the volume of the subpackaging tank is 50L, and the histiocyte resuspension liquid of every 500 chick embryos is injected into one subpackaging tank; the gauze bag type filtering device in the subpackaging tank comprises 18 layers of gauzes, and cell culture solution is supplemented to 50L scale marks of the subpackaging tank after injection and filtration of the histiocyte resuspension of the chick embryos are completed.

Preferably: in the step (10), the outlet of each sub-packaging tank is connected with a liquid distribution pipe with a plurality of liquid distribution heads, each liquid distribution head is connected to the injection port of one cell factory, and the cell suspension liquid of a plurality of cell factories is sub-packaged at one time.

Preferably: in the step (11), the temperature of a culture greenhouse of the cell factory is 38 ℃, and the culture time is 18-24 h.

Drawings

FIG. 1 is a microscopic image of viable cell count in the dispensing step of the present invention;

FIG. 2 is a microscopic image of cells in a cell factory 12 hours after the cell culture step of the present invention has been performed;

FIG. 3 is a microscopic image of cells in a cell factory 48h after the cell culture step of the present invention;

FIG. 4 is a microscopic image of the cells in the cell factory after 72h of the cell culture step of the present invention.

Detailed Description

In order to further understand the contents, features and effects of the present invention, the following embodiments are described in detail.

The method for preparing the chick embryo fibroblast in a large scale comprises the following steps,

(1) selecting chicken embryos and carrying out primary disinfection: selecting a plurality of SPF (specific pathogen free) chick embryos which are well developed in a chick embryo pre-incubation chamber according to embryos, sending the chick embryos into a disinfection chamber, disinfecting the surfaces of the chick embryos by using disinfectant spray, and then transferring the chick embryos into a greenhouse; in this step, selected SPF chick embryos are 9 days old, and the room temperature in the greenhouse is 38 ℃.

SPF refers to Specific Pathogen Free, SPF animals, i.e., Pathogen-Free animals, and refers to experimental animals that do not carry pathogens that are primarily potentially infectious or conditionally pathogenic and that interfere significantly with scientific experiments, except for pathogens that the clean animals should exclude. The SPF chick embryos selected in the step are chick embryos produced by SPF chickens (chick embryos which grow in a barrier system or an isolator and have no main chicken infectious disease pathogens which are particularly popular internationally and domestically) meeting the conditions. SPF chick embryos are sensitive to various pathogenic microorganisms, have good repeatability and are high-standard raw materials for developing various biological products such as human beings, poultry and the like.

In the specific operation of the step, the chick embryos are placed in a special tray, the tray is placed on a trolley, and the chick embryos which are selected and preliminarily sterilized are transferred into a greenhouse in a batch by the trolley. The disinfection operation is carried out in a special disinfection room, and the spraying and atomizing disinfection facility is adopted to carry out surface spraying and atomizing disinfection on the chick embryos. In this example, the number of selected SPF chick embryos for a single preparation is 2000.

(2) And (3) disinfecting the chick embryos again: taking out the chick embryo from the greenhouse, dipping iodine tincture by using a brush, disinfecting the upper part of the chick embryo, and deiodinating the chick embryo by using absolute ethyl alcohol after the iodine tincture is dried;

in the step, the chick embryos are taken out from the greenhouse, and the middle upper parts of the chick embryos are disinfected by iodine on the next day after being placed in the greenhouse. Specifically, dip in iodine tincture with the brush and disinfect to the part above the chick embryo central line, that is to say disinfect the well upper portion of chick embryo, the well upper portion of chick embryo is the position that the eggshell was opened in the follow-up, adopts iodine tincture disinfection can get rid of the germ in this position, avoids opening the eggshell when the germ gets into inside.

The iodine tincture has a strong disinfection effect in conventional disinfection liquid, and the iodine tincture is used for disinfection firstly, so that the certainty of the disinfection effect can be ensured. The absolute ethyl alcohol can be used for removing iodine (iodine can be dissolved in the absolute ethyl alcohol, so that the iodine is removed when the surface of the chick embryo is wiped), and can also be used for dewatering (the absolute ethyl alcohol absorbs water and then evaporates), so that the dryness of the surface of the chick embryo is ensured, the situation that liquid remained outside enters an air chamber when the air chamber of the chick embryo is opened in the subsequent process is effectively avoided, and the risk of pollution is reduced.

(3) Taking chicken embryos: opening the air chamber of the chick embryo, taking out the chick embryo, and putting the chick embryo into a plate;

the tools used in the step are mainly surgical skew scissors and skew tweezers, wherein the surgical skew scissors are used for opening eggshells and air chambers of the chick embryos, and the skew tweezers are used for taking the chick embryos out of the eggshells and the air chambers.

(4) Treating chicken embryos: clamping and removing the eyes of the chick embryo by using a tool;

the tools used in the step are mainly askew head tweezers, and the askew head tweezers are adopted to clamp the eyes of the chick embryo and remove the chick embryo.

In the invention, the traditional method is not adopted to remove the head, the limbs and the internal organs of the chick embryo, only eyeball removal treatment is carried out, and because tissues such as brain balls and internal organ tissues such as liver and the like are not digested by pancreatin, the quality of the chick embryo fibroblast preparation cannot be influenced, and the chick embryo fibroblast can be removed in one step in subsequent filtration. The benefits of removing the eyeball alone are three-fold, first: the working procedure of chicken embryo treatment is simplified, and about 80% of treatment time is saved; secondly, the preparation quantity of the fibroblasts of the unit chick embryo is improved, because a great amount of chick embryo skin is lost in the process of removing the head, the limbs and the internal organs, and each chick embryo can stably obtain 2.5-3 multiplied by 10 through the data statistics of the preparation method⁸The number of the chicken embryo cells is twice that of the traditional fibroblast preparation method; thirdly, because the treatment mode is simplified, the time of exposing the chick embryos to the external environment is reduced, and the risk of pollution is reduced.

(5) Cleaning chicken embryos: adding the chicken embryos into a cleaner, injecting the Hankel solution into the cleaner, shaking the chicken embryo cleaner to fully mix the chicken embryos with the Hankel solution, then sucking out the Hankel solution in the cleaner, and repeating the process of cleaning the chicken embryos for multiple times;

in the step, a plurality of the obtained chick embryos are subpackaged into a plurality of cleaners, and 2000 chick embryos are subpackaged into 7 chick embryo cleaners. The chick embryo purger is structurally including wasing the jar, is equipped with the tee bend lid at the top of wasing the jar, covers at the tee bend and is equipped with three interface, one for the inlet, one be extraction opening, another be the liquid outlet, be connected with feed liquor pipe and feed liquor pipe on the inlet and the lower extreme extends to the inner chamber upper portion of wasing the jar, is connected with the liquid suction pipe on the liquid outlet, and the lower extreme of liquid suction pipe extends to the inner chamber bottom of wasing the jar.

The multi-connection pipeline is adopted to connect the liquid inlet covered by the three-way on the top of each cleaner with the cleaning liquid storage tank, the multi-connection pipeline is adopted to connect the air suction port covered by the three-way on the top of each cleaner and install the vacuum pump on the multi-connection pipeline, and the multi-connection pipeline is adopted to connect the liquid outlet covered by the three-way on the top of each cleaner with the waste liquid storage tank and install the liquid pump on the multi-connection pipeline. Therefore, when cleaning liquid needs to be injected into the cleaning device, the vacuum pump is started, the vacuum pump extracts air in each cleaning tank through the air extraction opening, the internal air pressure is reduced, the cleaning liquid enters the cleaning tanks through the liquid inlet, the cleaning liquid stops after the chicken embryos are immersed in the cleaning liquid, the chicken embryo cleaning device is shaken to enable the chicken embryos to be fully mixed with the Hank's liquid, then the liquid extraction pump is started, and the Hank's liquid and the cleaning impurities are extracted from the bottom of each cleaning tank.

This chicken embryo purger washs the chicken embryo under relative airtight state, can guarantee the sterile condition of cleaning process, simultaneously through the multi-channel connection, two people once only can 7 purgers of concurrent operation, have saved the abluent time of chicken embryo greatly, have promoted the operating efficiency.

And repeating the processes of injecting, shaking and mixing the cleaning solution and extracting the cleaning solution, wherein the cleaning frequency is set to three times, and the thorough cleaning treatment of the chick embryos is realized.

(6) Digesting chicken embryos: pouring the cleaned chick embryos into a magnetic digestion bottle, stirring at a high rotating speed to completely break the chick embryos in the digestion bottle into pasty suspended tissues, then adjusting to a low rotating speed, stirring, adding pancreatin, and fully digesting the chick embryo tissues under the action of the pancreatin;

the magnetic digestion bottle, namely the magnetic stirring bottle in the step, is a stirring bottle taking magnetic stirring as a basic working principle, and comprises a base generating a periodically-changed magnetic action and stirring particles arranged in the bottle, wherein the stirring particles generate stirring action under the action of the magnetic force during working. The high rotating speed and the low rotating speed are relative, under the stirring action of the high rotating speed, the chick embryos are smashed to quickly form pasty suspension tissues, and the low rotating speed stirring has two functions: firstly, make the temperature of digestive juice more even, secondly under the stirring state, the histiocyte that embryo tissue outer layer has already digested drops to the digestive juice more fast in, makes the internal tissue expose in the digestive juice for the digestion progress, both saved the digestion time, obtains the cell that the vitality is strong again. In general, a high rotational speed means a rotational speed of 380r/min or more, and a low rotational speed means a rotational speed of 140r/min or less.

In the step, high-speed stirring and low-speed stirring are carried out at the temperature of 38 ℃, the duration time of the high-speed stirring is 5min, and the duration time of the low-speed stirring is 15 min; after stirring at a low rotating speed, removing a melting bottle to observe the sediment at the bottom, and taking the cerebro-sphere tissue, the liver, the vertebra bone, the leg part and the wing part bone in the sediment at the bottom which are clearly visible and no muscle tissue on the bone as the end point of the step of judging the digestion of the chick embryo.

In this step, the addition amount of pancreatin is: 10 ml/embryo.

(7) Tissue cell centrifugation: digesting the chicken embryo suspension tissue, adding serum into the digestive juice under the aseptic condition to neutralize pancreatin, and centrifuging by adopting a centrifuge;

in the step, the weight of the added serum is 3 percent of the total weight of the digestive juice; the rotation speed of the centrifuge is 3000r/min, and the centrifugation duration is 10 min.

(8) Resuspending the tissue cells: discarding the centrifuged supernatant, adding the cell growth liquid into the centrifuge tube, shaking the centrifuge tube to separate the histiocytes from the tube wall of the centrifuge tube, adding the histiocyte into the mixing bottle, and shaking the mixing bottle to make the histiocyte become suspension;

(9) filtering tissue cells: sucking the tissue cell resuspension into a subpackaging tank with a gauze bag type filtering device, filtering and intercepting undigested chick embryo tissues in the cell suspension by the cell suspension through the gauze bag type filtering device, and then supplementing a cell culture solution into the subpackaging tank;

in the step, the volume of the split charging tank is 50L, and the histiocyte resuspension of every 500 chick embryos is injected into one split charging tank; the gauze bag type filtering device in the subpackaging tank comprises 18 layers of gauzes, and after injection and filtration of the histiocyte resuspension of the chick embryos are completed, cell culture fluid is supplemented to the 50L scale mark position of the subpackaging tank, namely after injection and filtration operations are completed, the cell culture fluid is filled in the subpackaging tank.

(10) Subpackaging: subpackaging the cell suspension in the subpackaging tank to a plurality of cell factories;

viable cell counts may be performed prior to dispensing, and a microscopic image of viable cell counts is shown in fig. 1.

In this step, the outlet of each dispensing tank is connected with a dispensing tube with a plurality of dispensing heads, each dispensing head is connected to the injection port of one cell factory, and the dispensing of the cell suspension of a plurality of cell factories is performed at one time. Specifically, the cell factory is selected as a 10-layer structure cell factory, and the number of liquid separation heads on each liquid separation tube is 10, so that 10 cell factories are separately loaded at a time. Each cell factory was filled with about 2000ml of liquid, so 50L of cell suspension was co-filled into 25 cell factories.

(11) Cell culture: placing the cell factory into a greenhouse for static culture to obtain a monolayer chicken embryo fibroblast;

in the step, the temperature condition of a culture greenhouse of the cell factory is 38 ℃, and the culture time is 18-24 h.

Microscopic images of cells in the cell factories with culture times of 12h, 48h and 72h are given in fig. 2, 3 and 4, respectively, showing morphological changes of healthy cells over time.

Has the advantages that:

TABLE 1 comparison of efficiency and contamination risk

From a comparison of table 1 it can be seen that: the preparation of the chick embryo fibroblast is carried out by adopting the large-scale production mode, the operation steps are greatly simplified, meanwhile, the improved equipment is adopted, the production efficiency is greatly improved, the time of exposing the fibroblast preparation process to the outside is greatly shortened, and the risk of product pollution is reduced.

TABLE 2 comparison of Monoblast cell yield to cell viability

From table 2, it can be seen that the fibroblasts prepared by the two production methods have equivalent activities, while the mass production mode simplifies the chick embryo treatment mode, does not remove heads, limbs and viscera, improves the magnetic digestion mode, ensures that the tissue digestion is more sufficient, doubles the cell yield of single embryos, and greatly saves the production cost.

The aseptic operation of the large-scale production is guaranteed:

to realize the sterility of large-scale production, facilities are the foundation for ensuring the sterile environment in a large aspect, the GMP standardization is the software guarantee, and the personnel standard operation is the core. The success or failure of the continuous aseptic operation must be determined by details, and the following principles are grasped in details.

1. Ensuring that all materials entering the clean area are low in microbial limit and even sterile as far as possible, for example, chicken embryos enter the C-grade clean area from the D-grade pre-incubation area, all the chicken embryos need to be subjected to comprehensive spray sterilization through high-quality disinfectant, enter a local A-grade clean area below the B-grade clean area from the C-grade clean area, and are subjected to iodine disinfection and deiodination and dehydration of absolute ethyl alcohol. These aspects are all in the practice of the principle that "all material entering the clean zone is low microbial limit or even sterile".

2. The operation steps are simplified as much as possible, such as the chick embryo treatment stage, the chick embryo treatment in the traditional mode is to remove the head, the internal organs and the limbs, and the description is only to remove the eyeballs, and in terms of efficiency, only the eyeballs are removed, which is only 20% of the operation time in the traditional mode, and in terms of cost, a large amount of skin cells are discarded when the head, the limbs and the internal organs are removed together with the skin at the chest and abdomen.

3. The frequency and the time of exposing the sterile raw materials to the outside are reduced as much as possible, the preparation process of the cell nutrient solution and the subpackaging process of the cell nutrient solution are all pipelined, and the frequency and the time of exposing various liquids when the liquids are poured between bottles are reduced, so that the risk of pollution is reduced.

4. Human operation is reduced as much as possible, and equipment operation is used as much as possible, so that the probability of human error is reduced. Traditional chicken embryo washs, the majority carries out the washing of chicken embryo in the beaker, 1 time 2 personnel can only operate a beaker, the time of wasing the cost is long, the time that the chicken embryo exposes external environment is also long, we have carried out the improvement of chicken embryo cleaning instrument, add the chicken embryo in the chicken embryo purger, after connecting the pipeline, once only can operate 7 containers and wash, the production efficiency is greatly improved, the cleaning fluid circulation is carried out between equipment to the operation process is whole, artificial operation error has been reduced.