Sample preservation solution and method for obtaining large number of high-quality pig ear edge fibroblasts

Abstract

The invention provides a sample preservation solution and a method for obtaining a large number of high-quality pig ear edge fibroblasts, and belongs to the technical field of cell culture. According to the method, a place which is thin in ear part and avoids large blood vessels is selected for disinfection sampling, then the sample is transferred out of a colony house for treatment, pollution of fungi and bacteria to the sample can be reduced, tissue activity can be maintained for 7 days as long as the used sample preserving fluid, and cells obtained through separation are good in activity and normal in proliferation speed. Serum in the sample preservation solution can provide nutrition for tissues and maintain stability, and antibiotics can inhibit reproduction of bacteria and fungi. In the subculture process, the subculture ratio of the pig ear edge fibroblasts is adjusted to be 1: 2, the density is limited to be 70%-90%, and then subculture is conducted, so that the phenomenon that the cells are aged too early and the cell contact inhibition reaction is caused due to the fact that the cell density is too low or too high is avoided. During freezing preservation, the proportion of serum in the freezing liquid is increased, so that the pig ear margin fibroblasts are better preserved.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a sample preservation solution and a method for obtaining a large number of high-quality pig ear edge fibroblasts. Background Art

[0002] The significance of the isolation and culture of pig ear fibroblasts is to establish a somatic cell resource bank for precious pig breeds, to achieve long-term preservation of genetic materials, and to conduct medical experiments or live recovery when needed. When performing isolation and culture, the ear tissue of the pig needs to be collected first. After disinfection and cleaning, the tissue is placed in a sample preservation solution and transported back to the laboratory under low temperature conditions. The ear tissue is disinfected and cleaned in a clean bench, the skin tissue is separated and the dermis is retained. The dermis is cut into pieces and evenly spread in a cell culture dish. By adding a culture medium containing serum, the tissue block can adhere to the culture dish and remain active for a long time under the culture conditions of 37°C and 5% CO2, and the newly formed fibroblasts can swim out and grow near the attached tissue block. In order to maintain the stability of the cell genome and prevent cell aging, the cells are resuspended in a cell cryopreservation solution. At this time, the metabolic activity in the cell stagnates and enters a dormant state. It can be stored for a long time and revived when needed. Therefore, the activity of the sample tissue, the method of separating cells, the ratio of the freezing solution and the conditions for cell recovery are all key factors in obtaining high-quality cells.

[0003] Currently, the commonly used pig ear margin tissue sample preservation solutions are mainly physiological saline and DPBS. The activity of the sample tissue is short-lived, and cell isolation and culture are usually required within a short period of time after collection. In addition, the established pig ear margin fibroblast isolation and culture system has a high cell contamination rate, and the yield and viability of fibroblasts are both low. Summary of the invention

[0004] The purpose of the present invention is to provide a sample preservation solution and a method for obtaining a large number of high-quality pig ear edge fibroblasts. The sample preservation solution can keep the tissue active at a high level and extend the preservation time to more than 7 days. A large number of high-quality ear edge fibroblasts can be separated at any time within 7 days.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a sample preservation solution, which consists of 10 mL of DMEM, 500 μL of serum and 100 μL of 100× penicillin-streptomycin-amphotericin.

[0007] The present invention also provides a method for obtaining a large number of high-quality pig ear edge fibroblasts using the sample preservation solution, comprising the following steps:

[0008] (1) Tissue sampling: Select a thin part of the ear that is away from large blood vessels for disinfection, use ear forceps to take a sample about half the size of a fingernail from the edge of the pig's ear and put it into the sample preservation solution, and transfer it outside the pen; clean the longer hair on the collected sample and disinfect it, rinse the alcohol with Dulbecco's phosphate solution after disinfection, and then transfer it to Dulbecco's culture medium to obtain the ear tissue sample, which is stored at 4°C;

[0009] (2) Primary cell isolation and culture: Cut the ear tissue sample from step (1) into pieces with an area less than 1 mm. 2 , obtain tissue blocks, transfer the tissue blocks to a cell culture dish, evenly distribute them on the bottom of the dish, invert the cell culture dish for culture, and after the tissue blocks adhere to the wall, place the cell culture dish upright and add Dulbecco's medium for culture;

[0010] (3) Subculture: The tissue blocks in the primary cells of step (2) are removed, washed, and digested. After the cells become round and most of the cells are detached from the wall, the digestion is terminated with Dulbecco's medium, and the cells are centrifuged, resuspended, inoculated, and subcultured to obtain pig ear margin fibroblasts;

[0011] (4) Freezing: Discard the old culture medium in the cell culture dish, wash and digest, terminate the digestion with Dulbecco's medium and centrifuge, add freezing medium to resuspend the pig ear marginal fibroblasts, divide and freeze in a -80℃ refrigerator, and transfer to a nitrogen tank for long-term storage after freezing.

[0012] Furthermore, the temperature of the primary cell separation and culture in step (2) is 37° C., and the inverted culture time is 4 h to 6 h.

[0013] Furthermore, the condition for each subculture in step (3) is that the cell mixing degree reaches 70% to 90%, and the cell subculture ratio is 1:2.

[0014] Furthermore, the solution used for washing in step (3) is Dulbecco's phosphate solution, and the enzyme used for digestion is pancreatic enzyme.

[0015] Furthermore, the solution used for washing in step (4) is Dulbecco's phosphate solution, and the enzyme used for digestion is pancreatic enzyme.

[0016] Furthermore, the volume ratio of the components in the freezing solution in step (4) is DMEM: serum: DMSO = 7:2:1.

[0017] Beneficial effects of the present invention:

[0018] (1) The present invention prepares a sample preservation solution composed of 10 mL DMEM, 500 μL serum and 100 μL 100× penicillin-streptomycin-amphotericin. The serum in the sample preservation solution can provide nutrition to the tissue and maintain stability, and the antibiotics can inhibit the reproduction of bacteria and fungi. The sample preservation solution is used to preserve ear tissue, so that the tissue can maintain high activity, and the tissue activity can be maintained for up to 7 days. Within 7 days, ear edge fibroblasts with good activity and normal proliferation rate can be isolated at any time.

[0019] (2) The present invention selects a thinner part of the ear and avoids large blood vessels for disinfection sampling, and then transfers the sample to the outside of the pen for subsequent processing, which can effectively reduce the contamination of fungi and bacteria to the sample. The serum in the sample preservation solution used can provide nutrition to the tissue and maintain stability, and the antibiotics can inhibit the reproduction of bacteria and fungi. During subculture, since fibroblasts rely on high density growth, the present invention also adjusts the subculture ratio of pig ear edge fibroblasts to 1:2 to avoid the occurrence of premature cell aging due to too low inoculation density. In addition, since the cell density is too low to reach the number of cells required for subculture, and the cell density is too high, it will cause the cells to undergo contact inhibition reaction, resulting in an increase in the cell apoptosis rate. The present invention also limits the density to 70% to 90% before subculture. During cryopreservation, the proportion of serum in the freezing solution is increased to better preserve the pig ear edge fibroblasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 This is a diagram of an inverted culture dish in Example 1;

[0022] Figure 2 The fresh tissue and tissue preserved in the preservation solution in Example 2 and the trypan blue activity rate after being stored at 4 degrees for 7 days;

[0023] Figure 3 The cell growth curves of fresh tissue and tissue stored at 4 degrees for 7 days in Example 3;

[0024] Figure 4 This is the ability of pig ear marginal fibroblasts to migrate out of fresh tissue and tissue stored at 4 degrees for 7 days in Example 3. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described in detail below, and the embodiments are intended to explain the present invention and should not be construed as limiting the present invention. If specific techniques or conditions are not specified in the embodiments, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. If the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0026] Example 1

[0027] Isolation and culture of high-quality pig ear margin fibroblasts:

[0028] Preparation method of sample preservation solution:

[0029] 10mL DMEM + 500μL serum + 100μL 100× penicillin-streptomycin-amphotericin, mix well and filter sterilize using a 0.22μm filter. Do not sterilize by high pressure. Once the sample preservation solution is prepared, it should be stored at 4°C for no more than 3 weeks.

[0030] Preparation method of freezing solution: prepare cell freezing solution according to the ratio of DMEM: serum: DMSO = 7:2:1.

[0031] Tissue sampling method:

[0032] (1) Select a thin part of the ear that is away from large blood vessels for disinfection. Use ear forceps to take an ear tissue sample about half the size of a fingernail from the edge of the pig's ear and put it into the sample preservation solution. Then transfer it outside the pen for subsequent processing.

[0033] (2) Clean the longer hairs on the collected ear tissue samples and transfer them to a 15 mL centrifuge tube containing 75% alcohol. Shake and oscillate for 30 seconds. Use long forceps to remove the pig ear tissue samples, rinse the alcohol with Dulbecco's phosphate-free saline solution (DPBS), and then transfer them to Dulbecco's medium (DMEM).

[0034] (3) Use a marker to mark the cap and wall of the centrifuge tube, seal the mouth of the centrifuge tube with sealing film, and record the sample.

[0035] (4) Store the collected samples in a 4°C car refrigerator and immediately send them to the laboratory for cell isolation within 24 hours.

[0036] (5) Waste after sampling should be disposed of harmlessly in accordance with the provisions of the Medical Waste Management Regulations.

[0037] Primary cell isolation and culture:

[0038] (1) Use scissors to cut the ear tissue sample into pieces smaller than 1 mm. 2 , obtain tissue blocks, and use a Pasteur pipette to transfer the moist tissue blocks to a 60 mm cell culture dish so that they are evenly distributed on the bottom of the dish.

[0039] (2) Turn the culture dish upside down (see Figure 1 ), culture in a 37°C incubator, observe every 2 hours, wait until the culture medium is slightly dry (do not let the culture medium dry completely), and the tissue blocks adhere to the wall, then place the cell culture dish upright.

[0040] (3) Add 4 mL of Dulbecco's medium (DMEM) preheated at 37°C to the culture dish. After 12 hours, observe whether there is contamination. If there is contamination, deal with it in time (discard or wash it again). Observe the cell migration and growth status every 1 day. When all the cells have covered the bottom of the culture dish that is not covered by the tissue mass, subculture is performed.

[0041] (4) Mark cell treatment methods and cell status.

[0042] Subculture

[0043] (1) Observe the confluence of primary cells under a microscope. When the confluence reaches 80%, subculture is performed.

[0044] (2) Remove the tissue blocks from the primary cells in a clean bench, wash the cells 2-3 times with Dulbecco's phosphate-free saline solution (DPBS), add 1 mL of trypsin and place in an incubator for digestion for 3 min, remove the cells and observe the cell wall detachment under a microscope. When the cells become round and most of the cells are detached, immediately add 2 mL of Dulbecco's medium (DMEM) to terminate the digestion. Pipette the cell suspension into a 5 mL centrifuge tube and centrifuge at 1000 rpm for 5 min to keep the cell pellet.

[0045] (3) Resuspend the cell pellet in new Dulbecco's medium (DMEM), evenly inoculate the cells into two 60 mm cell culture dishes, label them as P1 ear margin fibroblasts, and mark the date and species. Place the cells in an incubator and change the medium every 2 days.

[0046] (4) When the cell confluence reaches 70% to 90%, the next subculture can be performed.

[0047] freezing:

[0048] (1) Discard the old culture medium in the cell culture dish, wash the cells once or twice with preheated Dulbecco's phosphate-buffered saline (DPBS), add trypsin to digest the cells, and centrifuge at 1000 rpm for 5 minutes after terminating the digestion.

[0049] (2) Discard the cell culture medium and add the pre-prepared freezing solution at room temperature to resuspend the cells and adjust the cell density to 1-5×10 6 / mL. Then divide into cryopreservation tubes, mark the cell generation, cell source and cryopreservation date. Put into the cell program freezing box and freeze in -80℃ refrigerator. After freezing for 10 hours, transfer the cells to liquid nitrogen tank for long-term storage.

[0050] Example 2

[0051] Six ear tissue samples were collected in the sample preservation solution according to step (1) of the tissue sampling method in Example 1. Three samples were placed in a 4-degree refrigerator for 7 days. The remaining three samples (NC group) were subjected to the remaining steps of the tissue sampling method in Example 1 and the primary cell separation and culture steps to obtain pig ear margin fibroblasts. The cell viability, growth curve, and ability to migrate out of the tissue were tested. The other three groups were taken out after being stored in a 4-degree refrigerator for 7 days, and the remaining steps of the tissue sampling method in Example 1 and the primary cell separation and culture steps were performed to obtain pig ear margin fibroblasts. The cell viability, growth curve, and ability to migrate out of the tissue were tested. The results are as follows: Figures 2 to 4 shown.

[0052] Depend on Figures 2 to 4 It can be seen that the sample preservation solution can maintain tissue activity for up to 7 days. After 7 days of tissue preservation, the cultured cells have no obvious differences in vitality, growth curve and ability to migrate out of the tissue compared with cells cultured from fresh tissue. The isolated cells have good activity and normal proliferation rate.

[0053] The present invention selects a thinner part of the ear and avoids large blood vessels for disinfection sampling, and then transfers the sample outside the pen for subsequent processing, which can effectively reduce the contamination of fungi and bacteria to the sample. The 5% serum contained in the sample preservation solution can provide nutrition for the tissue and maintain stability, which can not only ensure the activity of the tissue, but also will not cause waste of serum. The penicillin-streptomycin-amphotericin B contained in it can inhibit the contamination of tissues and cells caused by some Gram-negative bacteria, Gram-positive bacteria, fungi and yeast; the DMEM high-sugar culture medium contained in it can provide sufficient carbon sources and other nutrients for tissues and cells. The sample is placed in low-temperature storage and transportation, which can keep the sample at a high activity. The currently commonly used sampling method is to cut the ear edge tissue and directly put it into DPBS solution or physiological saline for low-temperature storage, which can neither keep the tissue block active for a long time, but also easily cause serious contamination of the tissue block, and it cannot be cleaned in the later stage.

[0054] During primary culture, the adhesion state of the tissue block is observed every 2 hours, and the cell culture fluid is added in time, so that the activity of the tissue block can be maintained for a long time, and the tissue block can be reused to release new fibroblasts. In addition, since fibroblasts rely on high-density growth, too low inoculation density will cause premature aging of cells. The present invention also adjusts the pig ear edge fibroblast passage ratio to 1:2, at which time the cell growth rate is faster, and the cell apoptosis rate and aging degree will not appear at a high level.

[0055] Furthermore, since too low a cell density will not reach the number of cells required for subculture, and too high a cell density will cause contact inhibition in the cells, leading to an increase in the rate of cell apoptosis, the present invention also adjusts the cell density during subculture to 70% to 90% before subculture.

[0056] The freezing solution of the present invention is prepared in advance and placed at room temperature for standby use, so that the heat generated by the temporary preparation can be prevented from damaging the cells. The cell freezing solution is configured according to the ratio of DMEM: serum: DMSO = 7:2:1, which can maximize the use of the nutrients in the preservation solution without causing waste of resources. At the same time, it can reduce cell damage and ensure that the cells have a high viability after recovery. Increasing the proportion of serum in the freezing solution can better preserve certain fragile cells and precious cells. Experiments have shown that the sample preservation solution can maintain tissue activity for up to 7 days, and the separated cells have good activity and normal proliferation rate.

[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A sample preservation solution, characterized in that: The sample preservation solution consists of 10 mL DMEM, 500 μL serum and 100 μL 100× penicillin-streptomycin-amphotericin.

2. A method for obtaining a large number of high-quality pig ear edge fibroblasts using the sample preservation solution of claim 1, characterized in that: The steps include: (1) Tissue sampling: Select a thin part of the ear that is away from large blood vessels for disinfection, use ear forceps to take a sample about half the size of a fingernail from the edge of the pig's ear and put it into the sample preservation solution, and transfer it outside the pen; clean the longer hair on the collected sample and disinfect it, rinse the alcohol with Dulbecco's phosphate solution after disinfection, and then transfer it to Dulbecco's culture medium to obtain the ear tissue sample, which is stored at 4°C; (2) Primary cell isolation and culture: Cut the ear tissue sample from step (1) into pieces with an area less than 1 mm. 2 , obtain tissue blocks, transfer the tissue blocks to a cell culture dish, evenly distribute them on the bottom of the dish, invert the cell culture dish for culture, and after the tissue blocks adhere to the wall, place the cell culture dish upright and add Dulbecco's medium for culture; (3) Subculture: The tissue blocks in the primary cells of step (2) are removed, washed, and digested. After the cells become round and most of the cells are detached from the wall, the digestion is terminated with Dulbecco's medium, and the cells are centrifuged, resuspended, inoculated, and subcultured to obtain pig ear margin fibroblasts; (4) Freezing: Discard the old culture medium in the cell culture dish, wash and digest, terminate the digestion with Dulbecco's medium and centrifuge, add freezing medium to resuspend the pig ear marginal fibroblasts, divide and freeze in a -80℃ refrigerator, and transfer to a nitrogen tank for long-term storage after freezing.

3. The method for obtaining a large number of high-quality pig ear edge fibroblasts according to claim 2, characterized in that: The temperature for the primary cell separation and culture in step (2) is 37° C., and the inverted culture time is 4 h to 6 h.

4. The method for obtaining a large number of high-quality pig ear edge fibroblasts according to claim 2, characterized in that: The condition for each subculture in step (3) is that the cell mixing degree reaches 70% to 90%, and the cell subculture ratio is 1:

2.

5. The method for obtaining a large number of high-quality pig ear edge fibroblasts according to claim 2, characterized in that: The solution used for washing in step (3) is Dulbecco's phosphate solution, and the enzyme used for digestion is pancreatic enzyme.

6. The method for obtaining a large number of high-quality pig ear edge fibroblasts according to claim 2, characterized in that: The solution used for washing in step (4) is Dulbecco's phosphate solution, and the enzyme used for digestion is pancreatic enzyme.

7. The method for obtaining a large number of high-quality pig ear edge fibroblasts according to claim 2, characterized in that: The volume ratio of each component in the freezing solution of step (4) is DMEM: serum: DMSO = 7:2:1.

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