Cell collection method for improving transmission electron microscope picture quality of in-vitro culture cells

Abstract

The invention relates to a cell collection method for improving transmission electron microscope picture quality of cells cultured in vitro, and belongs to the field of transmission electron microscope biological sample preparation. Comprising the following steps: (1) reviving cells: discarding a drug-containing culture solution, and adding a fresh complete culture solution; (2) scraping cells and adding glutaraldehyde stationary liquid for pre-immobilization; (3) pre-fixation: sucking the supernate by using a needle cylinder, and adding 1mL of stationary liquid along the tube wall; (4) rinsing: rinsing for 5 minutes by using 0.1 M PB and pure water; (5) carbonization: putting the cell agglomerates into a carbonization cabin, and fumigating and burning the cell agglomerates for 1-1.5 hours by using 1% osmium tetroxide; (6) pure acetone induction: putting the sample obtained in the step (5) into 100% acetone for 5 minutes twice; (7) permeating: adding a mixed solution of epoxy resin and 100% acetone, centrifuging, and putting into a 38 DEG C oven for 2 hours; and (8) embedding and curing. According to the method, the cells can be collected in a short time, the influence of operation conditions and environment on the collection process is reduced, and the TEM picture quality of the adherent cells cultured in vitro is improved.

Description

Technical Field

[0001] The invention relates to the technical field of transmission electron microscope biological sample processing and preparation, in particular to a method for collecting and preparing transmission electron microscope samples of adherent cells cultured in vitro. Background Art

[0002] In the more than 100 years since the successful in vitro generation of frog embryo nerve fibers in 1907, cell culture technology has been widely used in many fields of medicine. As a powerful supplement to limited in vivo experiments, the scientific research value of in vitro cultured cells is also being adopted and innovated by more and more researchers. In vitro cultured cells themselves have obvious advantages, with quantitative controllable culture conditions, strong growth capacity, simple operation, short cycle, and good repeatability. The disadvantages are poor representativeness, high cost, difficulty in simulating dynamic processes, and genetic variation. As the most basic culture technology, it has also been continuously optimized and improved. Transmission electron microscopy, as a powerful means of ultrastructural observation, has also been widely used in cell culture observation.

[0003] The ideal state for observation of cultured cells is in situ observation, that is, in situ fixation and embedding scheme. Although there are fewer steps, the operation details are difficult to grasp, and there are fewer cells. It needs to be repeated at multiple sites and sliced ​​from different parts to obtain a representative view of the structural characteristics of each cell sample. It is currently also used in optoelectronic combined experiments, which is conducive to the positioning and observation of organelles. At present, the most commonly used method in China is to enrich the cultured cells for observation. There are many cells, and different cell sections can complement each other, so more ultrastructural information can be obtained. The collection scheme mainly includes fixation first and then collection and collection first and then fixation. Experiments have shown that the two schemes have their own advantages and disadvantages. The former has a good fixation effect, and the cells are not easy to clump. The pre-treatment process is difficult, and the cells are easy to lose. It is even necessary to do pre-embedding of agar to convert the cell samples into tissue-like samples; the latter has the advantage that the cells are easy to clump, and the disadvantage is that the cells are easy to be poorly fixed or damaged.

[0004] The sample preparation process for transmission electron microscopy has many steps, and the focus is on the influence of the sampling process on the ultrastructure, which is crucial to the success or failure of the entire electron microscope experiment. In vitro cultured cells, due to the particularity of their living environment, have many factors that affect the quality of ultrastructure, including the state and activity of the cells, the operation techniques when sampling, and the collection method. Each laboratory has slightly different conditions, resulting in adverse consequences such as unclear structure, tissue dissolution, and microbial contamination. For example: 1. If there are many impurities in the cells, pour out the culture medium before collection and quickly wash with saline to remove impurities. If the rinsing time is too long, the cells will be hypoxic and lack of nutrition, and the ultrastructure will be abnormal or even edema; 2. If the cells are left to stand for too long before collection, the cells are in a "starved state" of lack of nutrition and hypoxia. At this time, the collected cells will also have a variety of illusions, such as increased autophagy. 3. The fixation effect is poor, or the fixative is unqualified, including unqualified concentration, unqualified pH value, etc., or the collection operation time is too long, causing tissue autolysis, etc. Summary of the invention

[0005] In order to overcome the above-mentioned defects of the existing in vitro culture cell collection scheme, the present invention provides a cell processing method for improving the quality of TEM images of adherent cells. This method concentrates the advantages of multiple cell collection schemes, effectively avoids the disadvantages, achieves full and effective fixation, and can obtain cell clumps of appropriate size. Combined with the carbonization sample preparation scheme, it not only eliminates the generation of osmium black particles at the source, but also plays a key role in improving the image quality, and can quickly prepare samples to meet the needs of different scientific research teams.

[0006] To achieve the above object, the technical solution adopted by the present invention is: A cell collection method for improving the quality of transmission electron microscope images of in vitro cultured cells comprises the following steps: (1) "Resurrection" of cells: Culture the cells until the density reaches 80%, discard all the drug-containing culture medium, add 1 mL of fresh complete culture medium, and place in a 37°C incubator for 5 min; (2) Scrape cells: Use a cell scraper to quickly push the cells in one direction, transfer them with a pipette, place the complete culture medium containing the cells in a 2 mL round-bottom EP tube, add 100 μL of glutaraldehyde fixative for pre-fixation, and centrifuge at 100-2500 rpm / min for 5-10 min; (3) Pre-fixation: remove the supernatant with a syringe, add 1 mL of glutaraldehyde fixative along the tube wall, and place in a 4°C refrigerator for 4 h; (4) Rinse: rinse once with 0.1 M PB for 15 min; then rinse twice with pure water for 5 min each time; (5) Carbonization: After rinsing, the cell mass is taken out and placed on a small piece of glass slide or a small bottom cup as a carrier. The excess liquid is absorbed with a pointed filter paper and placed in a carbonization cabin. Fumigation and burning with 1% osmium tetroxide is carried out for 1-1.5 h. (6) Pure acetone induction: The sample obtained in step (5) was placed in 100% acetone for 5 min, twice; (7) Infiltration: Add 1 mL of a mixture of epoxy resin and 100% acetone in a volume ratio of 1:1 to the sample induced in step (6), centrifuge at 1000 r / min for 5 min, and place in a 38°C oven for 2 h. Replace with pure epoxy resin for infiltration, cover the tissue with a small amount, and place in a 38°C oven for 1 h.

[0007] (8) Embedding and curing: embedding into a pure epoxy resin silicone plate and placing in an oven at 40°C for 1 h and 70°C for 10 h; After embedding and solidification, the cells were cut into ultrathin sections using an ultrathin microtome, and the cell structure was observed using a transmission electron microscope.

[0008] Furthermore, in steps (2) and (3), the mass concentration of the glutaraldehyde fixative is 2.5%, and the pH is 7.2-7.4.

[0009] Furthermore, in step (3), the cell clumps are controlled to be half the size of a mung bean before adding the glutaraldehyde fixative.

[0010] Furthermore, in step (4), the pH of PB is controlled at 7.2-7.4, and the pH of pure water is 7.0.

[0011] Furthermore, before step (1), the pH value of all reagents involved in the cell culture experiment needs to be measured to ensure that they meet the living needs of the cells; and comprehensive microbial testing is performed. On the basis of strict aseptic operation, a full range of microbial testing is performed, including the frozen cells themselves, all liquid reagents, and the cell room environment. Testing reagents must be used for testing to ensure that no microorganisms are detected in all items that come into contact with the cells.

[0012] Compared with the prior art, the present invention has the following beneficial effects: 1. The method of the present invention can collect cells in a short time, reduce the influence of operating conditions and environment on the collection process, and improve the quality of TEM images of adherent cells cultured in vitro.

[0013] 2. The "resurrected" cells in the method of the present invention can prevent the cells from being in a state of starvation and avoid the appearance of various false phenomena in the collected cells; at the same time, the collection time of the method of the present invention is short, which avoids the occurrence of cell tissue autolysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1TEM image of a normal group of human liver cells (L7702) collected in Comparative Example 1; Figure 2 The same samples collected in Example 1 Figure 1 TEM images of cells; Figure 3 TEM image of human neuroblastoma SHSY5Y cells collected for comparative example 1; Figure 4 Collected for Example 1 Figure 3 TEM images of cells in the same group; Figure 5 This is a TEM image of a normal group of bovine mammary gland cells collected in Comparative Example 2; Figure 6 Collected for Example 1 Figure 5 TEM images of cells in the same group; Figure 7 This is a TEM image of a normal group of mouse renal tubular epithelial TCMK-1 cells collected in Comparative Example 1; Figure 8 Collected for Example 1 Figure 7 TEM images of cells in the same group; Fig. 9 TEM image of normal group of human liver cells (L7702) collected in Comparative Example 2; Fig.10 Collected for Example 1 Fig. 9 TEM images of cells in the same group; Fig.11 Flow chart of the method of the present invention. DETAILED DESCRIPTION

[0015] The technical solution and effects of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0016] The common cell collection and processing scheme is shown in steps 1-4 in comparative example 1 and steps 1-5 in comparative example 2.

[0017] Comparative Example 1 The common cell collection and processing scheme of this comparative example is as follows.

[0018] 1. Pour out the culture medium in the culture bottle.

[0019] 2. Add an appropriate amount of 2.5% glutaraldehyde fixative and place in an ice bath for 3-5 min. The preparation of 2% glutaraldehyde fixative is prior art and will not be described in detail in the present invention. The amount of 2.5% glutaraldehyde fixative is 1-2 g of tissue per 100 mL.

[0020] 3. Use a rubber scraper to scrape the cells from the bottle wall, collect the fixative containing the cells in a 3 mL conical centrifuge tube, and centrifuge at 2000 r / min for 15-20 min to make the cells clump.

[0021] 4. After discarding the supernatant, slowly add 2.5% glutaraldehyde fixative to the cells along the wall of the tube. The amount of fixative added is 1 part of cells to 5 parts of fixative, and fix for 4 hours.

[0022] 5. Rinse: Rinse once with 0.1M PB at pH 7.2 for 15 min; then rinse twice with pure water at pH 7.0 for 5 min each time; 6. Carbonization: After rinsing, the cell mass is taken out and placed on a small piece of glass slide or a small bottom cup as a carrier. The excess liquid is absorbed with a sharp filter paper and placed in a carbonization cabin. Fumigation and burning with 1% osmium tetroxide for 1-1.5 hours; 7. Pure acetone induction: The sample obtained in step (5) was placed in 100% acetone for 5 min, twice; 8. Infiltration: Add 1 mL of a mixture of epoxy resin and 100% acetone in a volume ratio of 1:1 to the sample induced in step (6), centrifuge at 1000 r / min for 5 min, and place in a 38°C oven for 2 h. Replace with pure epoxy resin for infiltration, cover the tissue with a small amount, and place in a 38°C oven for 1 h.

[0023] 9. Embedding and curing: embed into pure epoxy resin silicone plate and place in oven at 40℃ for 1 h and 70℃ for 10 h.

[0024] After embedding and polymerization, the cells were cut into ultrathin sections using an ultrathin microtome, and the cell structure was observed using a transmission electron microscope.

[0025] Human liver cells (L7702), human neuroblastoma SHSY5Y cells, normal group of bovine mammary gland cells, and normal group of mouse renal tubular epithelial TCMK-1 cells were collected using the collection scheme of this comparative example, and their TEM images are shown in FIG. Figure 1 , 3 , as shown in 7. Figure 1 This is a TEM image of human liver cells (L7702) collected in comparative example 1. The cells are in a starved state, with increased autophagosomes in the cytoplasm, mitochondrial edema, and even vacuolization; Figure 3 This is a TEM image of human neuroblastoma SHSY5Y cells collected in Comparative Example 1. Due to the unqualified pH value of the fixative (the measured pH value of the fixative is 6.2), the cells are blurred and damaged as a whole, showing pyknotic changes; Figure 7 This is a TEM image of the normal group of mouse renal tubular epithelial TCMK-1 cells collected in Comparative Example 1. Due to factors such as inappropriate centrifugation speed (centrifugation rate 2000 rpm / min×15 min), the mitochondrial structure is blurred and edematous.

[0026] Comparative Example 2 The common cell collection and processing scheme of this comparative example is as follows.

[0027] 1. Pour out the culture medium in the culture bottle.

[0028] 2. Wash the cells once with sterile PBS, Hanks solution or serum-free culture medium to remove residual serum.

[0029] 3. Add a small amount of trypsin cell digestion solution (the trypsin digestion solution used in this comparative example contains 0.25% trypsin, EDTA and phenol red, and does not contain calcium and magnesium ions), just enough to cover the cells. The amount can be increased or decreased according to the efficiency of trypsin. Leave at room temperature for 30 seconds to 2 minutes (the digestion time of different cells is also different). When the bottle wall changes from translucent to transparent and dotted, or the wall of the culture bottle is seen to be white, the digestion can be stopped. Remove the trypsin cell digestion solution, add complete cell culture solution containing serum, and blow off the cells.

[0030] 4. Collect the cells in a 3 mL conical centrifuge tube and centrifuge at 2000-5000 r / min for 15-20 min to aggregate the cells.

[0031] 5. After discarding the supernatant, slowly add 2% glutaraldehyde fixative to the cells along the tube wall at a ratio of 1 part cell to 5 parts fixative, and fix for 4 hours.

[0032] 6. Rinse: Rinse once with 0.1M PB at pH 7.2 for 15 min; then rinse twice with pure water at pH 7.0 for 5 min each time; 7. Carbonization: After rinsing, the cell mass is taken out and placed on a small piece of glass slide or a small bottom cup as a carrier. The excess liquid is absorbed with a sharp filter paper and placed in a carbonization cabin. Fumigation and burning with 1% osmium tetroxide for 1-1.5 hours; 8. Pure acetone induction: The sample obtained in step (5) was placed in 100% acetone for 5 min, twice; 9. Infiltration: Add 1 mL of a mixture of epoxy resin and 100% acetone in a volume ratio of 1:1 to the sample induced in step (6), centrifuge at 1000 r / min for 5 min, and place in a 38°C oven for 2 h. Replace with pure epoxy resin for infiltration, cover the tissue with a small amount, and place in a 38°C oven for 1 h.

[0033] 10. Embedding and curing: embed into pure epoxy resin silicone plate and place in oven at 40℃ for 1 h and 70℃ for 10 h.

[0034] After embedding and polymerization, the cells were cut into ultrathin sections using an ultrathin microtome, and the cell structure was observed using a transmission electron microscope.

[0035] The collection scheme of this comparative example was used to collect the normal group of cow mammary cells and the normal group of human liver cells. Figure 5 , 9 As shown, Figure 5 This is a TEM image of a normal group of bovine mammary cells collected in Comparative Example 2. The sample was obtained after trypsin digestion, and the cell ultrastructure was blurred and destroyed, with obvious loss of ultrastructure details. Fig. 9 This is the TEM image of the normal group of human liver cells collected for comparative example 2. Due to the large cell clumps (about the size of soybeans), fixation, dehydration and penetration are poor, resulting in the appearance of the "knife sign", and the mitochondrial structure is blurred and edematous.

[0036] Example 1 The process of the cell collection method for improving the quality of transmission electron microscopy images of in vitro cultured cells of the present invention is as follows: Fig.11 The specific steps are as follows.

[0037] 1. Determine the pH value of the water used and adjust it according to the growth requirements of different cells. When testing the pH value, cut the pH test paper into small pieces and use a dropper to drop a large drop of the liquid to be tested on it. It is in an oversaturated state and the pH value is read within half a second. If it is acidic, add a small amount of NaOH to correct it. If it is alkaline, replace it with other brands of pure water. Do not use HCl to adjust the pH value.

[0038] This example collects normal groups of human liver cells (L7702), human neuroblastoma SHSY5Y cells, normal groups of cow mammary gland cells, and normal groups of mouse renal tubular epithelial TCMK-1 cells, which need to adjust the pH of water to 7.0-7.2 respectively. The cells here are all cells without special environmental requirements.

[0039] 2. Determine the pH value of the reagents used. Due to environmental influences, the pH of many preparations has a large deviation, so we need to check the pH of the cell culture medium. The pH value of the initial culture medium needs to be corrected and controlled between 7.0-7.4. For DMEM culture medium without FBS, use precision pH test paper to test the pH value. If the pH value is acidic, correct the pH value by adding 30 mL of 0.2 M disodium hydrogen phosphate per 100 mL of culture medium; 10×PBS buffer should not use commercial solutions. Experimenters should prepare it according to requirements and use it fresh. Note that when preparing PBS, solution A and solution B should be prepared separately and then mixed. After preparation, the pH value needs to be tested in the required range; when using 0.2M PB to prepare glutaraldehyde with a mass concentration of 2.5%, the pH value of the glutaraldehyde working solution should also be tested to ensure that it is within the effective range.

[0040] In this embodiment, the cells involved are all cells without special environmental requirements, so the pH value of the initial culture medium is controlled between 7.0-7.4; the pH value of the DMEM culture medium without FBS is controlled between 7.0-7.4; the pH value of the 10×PBS buffer is between 7.0-7.4; and the pH value of the glutaraldehyde fixative is controlled between 7.0-7.4.

[0041] 3. Comprehensively test microorganisms. On the basis of strict aseptic operation, all-round microbial testing should be carried out, including the frozen cells themselves, all liquid reagents, and the cell room environment. Testing reagents must be used for testing to ensure that no microorganisms are detected in all items that come into contact with the cells.

[0042] 4. "Resurrection" of cells: Culture the cells until the density reaches 80%, discard all the drug-containing culture medium, add 1 mL of fresh complete culture medium, and place in a 37°C incubator for 5 min; replace the culture medium with a new one when adding drugs to the cells to prevent the cells from starving and causing poor condition, which is called "resurrection cells."

[0043] The complete culture medium of each cell line used in this example is as follows: Human hepatocytes HL-7702 (L-02): RPMI-1640 high glucose medium plus 10% fetal bovine serum plus 1% penicillin-streptomycin, pH 7.2; Human neuroblastoma SH-SY5Y cells: DMEM / F12 (1:1) basal medium plus 10% fetal bovine serum plus 1% penicillin-streptomycin, pH 7.2; Bovine mammary epithelial cells were cultured in DMEM / F12 (1:1) basal medium supplemented with 10% fetal bovine serum, 10 ng / mL epidermal growth factor (EGF), 5 µg / mL insulin, 1 µg / mL epinephrine, 5 µg / mL transferrin, and 6.7 ng / mL selenate, pH 7.2; Mouse renal tubular epithelial TCMK-1 cells were cultured in DMEM basal medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, pH 7.2.

[0044] 5. Scrape cells: Use a soft cell scraper to quickly push the cells in one direction so that the cells are in an obtuse space and will not be squeezed and damaged by the angle. Use a pipette to transfer the cells. Place the complete culture medium containing the cells in a 2 mL round-bottom EP tube and add 100 μL of pH-standard glutaraldehyde fixative for pre-fixation. Centrifuge at 100-2500 rpm / min for 5-10 min (adjust the speed and time according to the cells). Cell clumps must be fixed in glutaraldehyde within 1 min.

[0045] In this example, normal groups of human hepatocytes (L7702), human neuroblastoma SHSY5Y cells, normal groups of bovine mammary gland cells, and normal groups of mouse renal tubular epithelial TCMK-1 cells were collected, and the centrifugation conditions were 1500 rpm / min×8min, 1000 rpm / min×6min, 1000 rpm / min×5min, and 2000 rpm / min×5min, respectively.

[0046] 6. Pre-fixation: Use a syringe to remove the supernatant, add 1 mL of glutaraldehyde fixative along the tube wall, and place in a 4°C refrigerator for 4 h. The size of the cell mass should be half the size of a mung bean. If the number of cells is too small and not clumped, they need to be pre-embedded with agar or the like; if the number of cells is too large, poor fixation will occur, ultrastructural details will be lost, or "knife-cutting signs" will appear. When the number of cells is large, the upper layer generally has more cell fragments, and the lower layer cells are better. The upper layer cells can be shoveled off, and the bottom cells can be retained before adding fixative; or a toothpick / needle can be used to pierce the center of the cell mass until the bottom of the cell to facilitate complete penetration of the fixative and achieve a full fixation effect.

[0047] 7. Rinse: Rinse once with 0.1M PB at pH 7.2 for 15 min; then rinse twice with pure water at pH 7.0 for 5 min each time.

[0048] 8. Carbonization: After rinsing, take out the cell mass and place it on a small piece of glass slide or a small bottom cup as a carrier. Use pointed filter paper to absorb excess liquid and place it in a carbonization cabin. Fumigate and burn with 1% osmium tetroxide for 1-1.5 hours.

[0049] 9. Pure acetone induction: The sample is placed in 100% acetone for 5 min, twice. After the first induction, remove as much acetone as possible from the tissue. The second induction requires about 100 µL of acetone to remain.

[0050] 10. Infiltration: Add 1 mL of a 1:1 mixture of 812 epoxy resin and 100% acetone to the sample induced in step 9, centrifuge at 1000 r / min for 5 min, and place in a 38°C oven for 2 h. Replace with pure epoxy resin for infiltration, cover the tissue with a small amount, and place in a 38°C oven for 1 h.

[0051] 11. Embedding and curing: embed into pure 812 epoxy resin silicone plate and place in oven at 40℃ for 1 h, 70℃ for 10 h (overnight).

[0052] After embedding and solidification, the cells were cut into ultrathin sections using an ultrathin microtome, and the cell structure was observed using a transmission electron microscope.

[0053] The collection scheme of this embodiment was used to collect normal groups of human liver cells (L7702), human neuroblastoma SHSY5Y cells, normal groups of cow mammary cells, and normal groups of mouse renal tubular epithelial TCMK-1 cells, and their TEM images are shown in FIG. Figure 2 , 4 , 6, 8, and 10. Figure 2 The same samples collected for this embodiment Figure 1 TEM image of cells. After the cells were “revived” before collection, the autophagosomes in the cells were reduced, and the mitochondrial membrane and cristae structures were clear and complete. Figure 4 Collected for this example Figure 3 TEM images of the same group of cells, with the pH of the aqueous reagent and culture medium adjusted to the appropriate range, show that the structures of each organelle are clear and complete, with no structural damage; Figure 6 Collected for this example Figure 5 TEM images of cells in the same group, using cell scraping as described in the protocol, show that the intracellular organelle structures are intact and clear, with no structural dissolution; Figure 8 Collected for this example Figure 7 TEM images of cells in the same group, collected according to the protocol after adjusting the centrifugation speed, showed that the organelle structure was clear and intact, with no obvious edema; Fig.10 Collected for this example Fig. 9 TEM images of the same group of cells, after removing the surface of the cell clumps and reducing the clumps, were collected according to the protocol, indicating that there was no "knife sign" and the organelle structure was clear and complete.

[0054] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A cell collection method for improving the quality of transmission electron microscopy images of in vitro cultured cells, characterized in that: The following steps are involved: (1) "Resurrection" of cells: Culture the cells until the density reaches 80%, discard all the drug-containing culture medium, add 1 mL of fresh complete culture medium, and place in a 37°C incubator for 5 min; (2) Scrape cells: Use a cell scraper to quickly push the cells in one direction, transfer them with a pipette, place the complete culture medium containing the cells in a 2 mL round-bottom EP tube, add 100 μL of glutaraldehyde fixative for pre-fixation, and centrifuge at 100-2500 rpm / min for 5-10 min; (3) Pre-fixation: remove the supernatant with a syringe, add 1 mL of glutaraldehyde fixative along the tube wall, and place in a 4°C refrigerator for 4 h; (4) Rinse: rinse once with 0.1 M PB for 15 min; then rinse twice with pure water for 5 min each time; (5) Carbonization: After rinsing, the cell mass is taken out and placed on a small piece of glass slide or a small bottom cup as a carrier. The excess liquid is absorbed with a pointed filter paper and placed in a carbonization cabin. Fumigation and burning with 1% osmium tetroxide is carried out for 1-1.5 h. (6) Pure acetone induction: The sample obtained in step (5) was placed in 100% acetone for 5 min, twice; (7) Infiltration: Add 1 mL of a mixture of epoxy resin and 100% acetone in a volume ratio of 1:1 to the sample induced in step (6), centrifuge at 1000 r / min for 5 min, and place in a 38°C oven for 2 h. Replace with pure epoxy resin for infiltration and cover the tissue with a small amount, and place in a 38°C oven for 1 h. (8) Embedding and curing: embedding into a pure epoxy resin silicone plate and placing in an oven at 40°C for 1 h and 70°C for 10 h; After embedding and solidification, the cells were cut into ultrathin sections using an ultrathin microtome, and the cell structure was observed using a transmission electron microscope.

2. The cell collection method for improving the quality of transmission electron microscopy images of in vitro cultured cells according to claim 1, characterized in that: The mass concentration of the glutaraldehyde fixative in steps (2) and (3) is 2.5%, and the pH is 7.2-7.

4.

3. The cell collection method for improving the quality of transmission electron microscopy images of in vitro cultured cells according to claim 1, characterized in that: In the step (3), the cell clumps are controlled to be half the size of a mung bean before adding the glutaraldehyde fixative.

4. The cell collection method for improving the quality of transmission electron microscopy images of in vitro cultured cells according to claim 1, characterized in that: In step (4), the pH of PB is controlled at 7.2-7.4, and the pH of pure water is 7.

0.

5. The cell collection method for improving the quality of transmission electron microscopy images of in vitro cultured cells according to claim 1, characterized in that: Prior to step (1), the pH value of all reagents involved in the cell culture experiment needs to be measured to ensure that they meet the living requirements of the cells; and comprehensive microbial testing is performed. On the basis of strict aseptic operation, a full range of microbial testing is performed, including the frozen cells themselves, all liquid reagents, and the cell room environment. Testing reagents must be used to ensure that all items in contact with the cells are free of microorganisms.

Images