细胞培养实验电子版本

Practical approach of animal cell cultureBASIC CELL CULTURE PROTOCOLS1. introductionThe culture of animal cells and tissues is now a widely used technique in many different disciplines from the basic sciences of cell and molecular biology to the rapidly evolving applied field of biotechnology. An introduction to the basic procedures is available in many laboratories and frequently features as integral part of undergraduate study in the biological sciences. In general, tissues culture procedures arc simple but require extreme care to avoid contamination. In this pamphlet, we introduce general procedures of cell culture and related techniques of biology. For a detailed and more comprehensive treatment of the subject, the reader may refer to the references.2.Instrumentation1. A Laminar Airflow CabinetFor media preparation, or when handling non-primate cell lines, culture manipulations can he conducted in a small front-opening cabinet. The cabinets are relatively cheap and have equipped with an ultraviolet (UV) light source to prevent contamination of the cabinet surface when not in use hut may not have an internal airflow. Good technique will ensure that this system does not offer operator protection against possible pathogens.If human or other primate cells are used then you require some protection against the possibility of transmission of infectious agents. For this purpose, most cell culture laboratories have an open-fronted laminar flow cabinet, which offers a space containing a vertical flow of filtered air and a horizontal working surface that can be disinfected. Air is drawn into the sterile working area of the cabinet through a high-efficiency particulate air (IIEPA) filter and 70-80%of the air is re-circulated to form an air curtain, which serves to maintain sterile operation and protect the operator. The exhausted air is also forced through a l-IFPA filter. The design is such that there is free access for the operator’s hands but a Perspex cover prevents the operator breathing over the working surface. The cabinet should be located in a corner of the laboratory free from draughts and air movement.The most common[y used cabinet for cell culture is 4fl long and designated as Class ii. The classification is a measure of the biological safety. A Class 11 cabinet is suitable for work with low to moderate toxic or infectious agents.2. IncubatorA water-jacketed (水套) CO2 incubator is generally used to maintain cultures at an optimal temperature and pH for cell growth. The water jacket provides a constant temperature environment with minimal danger of local heating effects in the inner chamber. Carbon dioxide is supplied from a gas cylinder into the optimal p11of the incubator The purpose of this is to maintain the optimal pH of the cultures by the bicarbonate-CO2 buffering system. The desired level of CO2 in the incubator is 5--I 0%, which can he maintained by a controller and gas analyzer(infra-red (红外线) for modern incubators). A water tray is positioned at the bottom of each chamber so that a relatively high Level of humidity can he maintained. This prevents excessive evaporation from the Culture vessels.3. An inverted microscope equipped with phase-contrast optics.4. Refrigerators (-20℃-80℃)Usually a domestic item will be found to be quite efficient and cheaper than special laboratory equipment. While auto-defrost freezers may be bad for some reagents (enzyme, antibodies, ctc.), they arc useful for most tissue culture stocks where their bulk and nature precludes severe cryogenic damage.5. Liquid nitrogen storageA facility for maintaining cell stocks is required unless all cultures are established from primary tissue. Cells are stored in plastic vials, which arc lowered into liquid nitrogen for long-term storage. Cryogenic plastic vials have strong seals to prevent leakage, which could result from large temperature fluctuations. The liquid nitrogen reservoir needs to he replenished at regular intervals. It is high[y recommended that a liquid nitrogen level indicator with alarm is used. This lowers the risk of damaging important cell stocks by inadvertently allowing the liquid nitrogen level to drop too low.6. Culture flasksPre-sterilized plastic flasks suitable for cell culture arc commercially available and are used by most laboratories. The plastic is sulphonated polystyrene, produced so that the surface is amenable for cell attachment and growth. The flasks are sterilized by y -irradiation and are suitable for single use.In the post, culture flasks were made of borosilicated glass. The flasks were re-cycled and required washing and autoclaving before use-a process, which could give, rise to contamination. The use of pre- sterilized and disposable plastic flasks has significantly reduced any contamination arising from the culture vessels.The most popular firms of plastic culture containers are multi-well plates. Petri dishes, and flasks (usually referred to as Tissue culture flasks or T-flasks). The multi-well plates can accommodate many replicates oi small volume cultures. The 24-well plates hold 3 ml per well and are well suited for cell growth experiments, for example to test for toxicity or stimulatory activity. The 96-well plates hold a volume of 0.3 ml per well and are suitable for cloning. Rapid dispensing of solutions into the 96-well plate is made easy by use of a multi-well pipettor.The Petri dishes and T-flasks can accommodate cultures of 2-100 ml and are suitable for both anchorage-dependent and suspension cells. The T-flasks are designated by the surface area available for cellattachment. A canted (angled) neck is provided so that sterile manipulation is easy. It is important to allowequilibrium to develop between the gas phase of the flask and the atmosphere of the incubator chamber, The l>flask caps can be adjusted to fit loosely. You should close the caps tightly once the flasks arc removed fromthe incubator.3. Materials3.1 Balanced Salt SolutionsA balanced salt solution (BSS) is composed of inorganic salts, usually including sodium bicarbonate, and is supplemented with glucose, although glucose and bicarbonate are often omitted. The compositions of some common examples are given in Table 3.1.Balanced Salt Solution a(g/L)Component Hanks Earle Dulbecco (PBS) GeyNaCl 8.00 6.8 8.0 7.0KCl 0.4 0.4 0.2 0.31MgCl2.6H2O 0.1e0.21MgSO4.7H2O 0.2 0.2Na2HPO4.12H2O 0.67 3.17KH2PO40.06 0.2CaCl2(anhyd.)b0.14 0.2 0.1e0.17NaHCO3c0.35 0.2 2.27D-glucose 1.0 1.0 1.0Phenol red d0.02 0.02Note: a. Dissolve each constituent separately uses sterile deionized distilled sterilize by autoclaving.b. Adding CaC½ last, and make up to literc. Adjust to PH 7.2 with 5.6% NaHC03 before using.d. Adding 5 mL/L, of 0.4% Phenol red.e. For washing, these components can’t be added.3.2 Culture MediumCells are cultured in a chemically complex liquid medium suitable for supporting growth for several generations. There are many standard media formulations, which have been developed for the growth of particular cell types.To determine which media to use for a specific cell line, it is advisable to search the literature to see if previous experiments suggest the use of a particular media formulation. Alternations may he performed. some media, such as Dulbecco’s modified Eagles medium (DMEM). have high concentrations of amino acids andvitamins and are suitable for prolonged cell growth. Other media, such as Ham’s F-12, contain a wide range of different components which may be required to satisfy the fastidious requirements of some cell lines. Combinations of standard formulations can also be used for cell growth. For example. a 1:1 v/v mixture of I)MEM and Hams F-12 has been found effective as a good basal medium for serum-free formulations to support the growth of a number of cell lines.Media can he supplied commercially as a I/liquid ready for use or as a liquid or powdered concentrate. The concentrates are much cheaper and are advisable if it is intended to List large volumes of one type of medium. The liquid concentrate (normally TO 7) is provided sterile and requires dilution with pre-sterilized distilled water Powdered medium should he dissolved in water and sterilized by filtration through a 0.22um filter. Filtration can be mediated by a vacuum pump or a peristaltic pump using tor double distillation. There are some unstable components (e.g. glutamine, bicarbonate) in culture medium and so complete liquid medium should not be autoclaved or stored for any length of time. Liquid medium can he purchased free of glutamine or bicarbonate. In this condition it can be stored at 4~C until required.Table 3.1 Commonly used culture mediumMedia CommentsBME Eagle’s basal medium; originally designed for mouse L and HeLa cellsMEM Eagle’s minimal essential medium; used for a wide variety of cell lines.I)MEM Dulbeeoo’s modification of Eagle’s minimal essential medium Has 4X the amino acid andvitamin concentration of MBM.PRMI1640Roswell Park Memorial Institute medium; Used for lymphocyte and hybridoma culturesHam’s F-l2Has a complex composition and is used for a variety of cell linesM199An extremely complex medium (61 components) which can support cell growth without serum3.3 Medium Supplement3.2.1 SerumSerum is normally added to culture media at a concentration of 10%(v/v) to promote cell growth. Cow (bovine) or horse (equine) serum is most commonly used, with fetal calf serum (FCS) being considered particularly effective because of its high content of embryonic growth factors. Table 3.2 lists some of the characteristics of bovine serum.Table 3.2 .1 Typical characteristics of bovine serumpH Osmolarity(mOsm/lProtein content() mg/mlAlbumin content(mg/ml))6.85-7.05 250-295 60-80 30-50Serum is purchased for laboratory use in 100 or 500m1 bottles from a cell culture supplier who will have treated the serum in a particular way. Typically, serum is sterilized by filtration through at least one 0.1 urn filter. Each batch is then tested a variety of microbial and chemical contaminants, including bacteria, fungi, and specific viruses, depending upon the source of the serum.Special treatment of serum offered by suppliers may include the following:• γ-Irradiation involves exposure of the serum h radiation from a 60Co source.• Heat inactivation involves incubation at 56℃for 30 min.• γ-Globulin removal involves ethanol fractionation which reduces the γ--Globulin content of the serum.This is important for monoclonal antibody production from hybridoma ) cultures because γ-Globulin derived from serum could interfere with the extraction or assay of the antibody.• Dialysis usually involves serum dialysis against 0.15 M NRA. This reduces the low molecular weight components of the serum but maintains its osmolarity (渗透压). This treatment eliminates the amino acid, carbohydrate, or nucleotide content of serum and might be required, for example, in a study of nutrient utilization of cells in culture. FCS is the most widely used media supplement. However, it is expensive and often in short supply. The serum is generally tested for performance and cytotoxicity. Performance is measured by the ability of the serum to support high plating efficiency and cell growth. The specifics of these tests are given by each manufacturer3.2.2 AntibioticsAntibiotics are often included in media for short-term cultures in order to reduce the risk of contamination. The optimal concentration of antibiotics should he determined empirically bearing in mind that hey may be cytotoxic. Antibiotics are often used in combination in culture medium, and the following cocktail can be recommended for general use:•Penicillin G (100U/ml) to inhibit the growth of Gram-positive bacteria.•Streptomycin (100mg /ml)to inhibit the growth of many Gram-negative and Gram-positive bacteria. •Amphotericin B (25 ug/ml ) as an anti-fungal agent.However, the use of antibiotics for routine sub-culture or in stock cultures should be discouraged because low levels of bacteria] or fungal contamination maybe masked and may cause problems at a later date. Furthermore, extensive use of antibiotics may cause the selective retention of antibiotic-resistant contaminants, which can cause future problem.3.23 Glutamine(谷胺酰胺)Glutamine 200 mML-glutamine 2.92 g Hank’s BSS 100mlDissolve glutamine in BSS and sterilize by filtration. Aliquots and store at -20℃3.2.4 TrypsinTrypsin is available as crude (Difco 1:250). Crude trypsins contain several other proteases which may he important in cell dissociation but may also he harmful to more sensitive cells. The usual practice is to use crude trypsin unless cell damage reduces viability or reduced growth is observed, when purified trypsin may he used. Pure trypsin has a higher specific activity and should therefore be used at a proportionally lower concentration, e.g., 0.05%.Trypsin solution (1.25%)Trypsin (1:250) 0.25 g PBS 100ml Sterilize by filtration. Aliqutos and store at 20℃4. General Technique for Tissue Culture4.1 SterilizationSolution, glassware, and other items can be sterilized by moist heat in an autoclave. Sterilize for 30 mm at a pressure of 15 lb/in24.2 Cell counting and monitoring4.2.1 Procedure for counting cells using the Dye-exclusion Haemocytometer ProcedureThis method consists of microscopically counting the number of cell in a very small volume of cell suspension. The Haemocytometer is a microscope slide with grids on it to give nine large squares divided by triple lines. Each large square has an area of I mm2. The depth of fluid in the slide chamber is 0.1 mm, and therefore the total volume of fluid over each large square is 0.1 mm3. Cells can also he counted in an electronic cell counter such as the Coulter counter.Trypan blue is often added to the cell suspension before counting. The dye penetrates the membrane of non-viable cells which arc stained blue and which can therefore he distinguished from viable cells. Viability gives an indication of the ability of a cell to divide.These haemocytometer-counting methods are simple and effective but can he laborious if many samples are being analyzed.Materials and equipments:1. 0.4% Ttypan bluc in HBSS: 0.4 g Trypan blue in l00ml HBSS, pass through a 0.22 um filter to remove any debris. Store at 4℃2. Haemocytometer with covetslip3. Hand-held counter4. Microscope— low power, X40 to X100 magnificationsSteps:1. Thoroughly clean the haemocytometer and covet-slip and wipe both with 70% alcohol before use.2. Cover the Neubauer chamber with a precision ground coverslip and press gently until Newton rings are visible. Place a drop of the cell suspension (usually diluted 1:3; 0.lml of cell plus 0.3 ml of 0.4% Thypan blue) at the edge of the coverslip.3. Place the chamber on the stage of an inverted microscope and count all the cells that exclude trypan blue in the four large squares in each corner of the central area. Count cells touching the right and upper lines hut not those touching the left and bottom lines. Nonviable cells absorb the dye and appear blue.4. It is essential to examine the cells within 5 mm of immersing them in Trypan blue.To calculate the viable divide the number of viable cells by the total number of cells that is viable and dead. Healthy cultures should contain more than 90% viable cells.Notes:The following can cause Inaccuracy of the haemocytometer method by affecting the volume of the chamber:• Overflowing the chamber and allowing sample to run into the channels• Incompletely filling the chamber• Air bubbles or debris in the chamber4.2.2 Indirect methods of cell determinationTetrazolium assay (MTT-Cell Proliferation)Methods for cell number determination in vitro are required for many different biological assays. The MTT-cell proliferation assay is a quantitative colorimetric assay for measurements of cellular proliferation, viability, and Cytotoxicity. The assay is based on cleavage of the yellow tetrazolium salt, MTT, which forms water-insoluble, dark blue formazan crystals. This cleavage only takes place in living cells by the mitochondria enzyme succinate-dehydrogenase. The water-insoluble formazan can he solubilized using isopropanol or another organic solvent (DMSO). The optical density of the dissolved material is measured spectrophotometrically; yielding absorbance as a unction of concentration of converted dye, which directly correlates to the number of metabolically active cells in the culture. The assay is simple, and well suited for analysis of large number of samples using 96-well microtier plates and a scanning multiwell spectrophotometer.Materials and equipments• MTT stock solution (5mg/ml): To make 10ml, add 50mg of MTT to PBS, pH7.5, and filterthrough a 0.22-um filter to sterilized and remove the small amount of insoluble residue.• DM50• 96-Well microtitre plate• Micro- E[ISA readerSteps:1.After incubation of cells for the chosen of time, add 20u1/well of the MTT solution (5mg/ml). Incubate the cells for additional 4 hr at 37℃and 5% CO2.2. Discard the medium from the wells and add 200ul DMSO to dissolve the formazan crystals. Measure the color intensity at 570nm (reference filter, 690nm) using a Micro-El ISA reader. For calculating the final cell number, absorbance values are compared with a standard curve for the cell line in question.4.3 Changing the medium and SubcultureChanging the mediumSteps:1. Examine culture carefully for signs of contamination or deterioration.2. Check the criteria of p1], cell density, or concentration and, based on your knowledge of the behavior of the culture, decide whether or not to replace the medium. If feeding is required, proceed as followings.3. Take the flask to sterile work area, remove and discard medium.4.Add same volume of fresh medium, prewarmed to 36.5℃5. Return culture to incubator.6. N ote: when a culture is at a low density and growing slowly, it may he preferable to “half-feed”. In this case, remove only half the medium at step 3 and replace it in step 4 with the same volume as was removed, SubcultureMaterials:Pipettes (sterile), medium (sterile), PBS (sterile). 0.25% trypsin in PBSA, EDTASteps:1. Withdraw medium and discard.2.Add PBS prewash to the side of the flask opposite the cells, so as to avoid dislodging cells, rinse the cells, and discard rinse. This step is designed to remove traces of serum, which would inhibit the action of the trypsin.3.Add trypsin to the side of the flask opposite the cells. Turn the flask over to cover the monolayer completely. Leave 15-30s and withdraw the trypsin, making sure beforehand that the monolayer has not detached.4. Incubate until cells round up.5. Add medium (3ml/ 25cm2) and disperse cells by repeated pipetting over the surface bearing the monolayer. Finally, pipette the cell suspension up and down a few times, with the tip of the pipette resting on the bottom corner of the bottle.6. Counting cells by hemocytometer.7. Dilute to appropriate seeding concentration by simply adding the cell suspension to the total volume of medium required for distribution to fresh culture flasks. This ensures that each flask will contain the same concentration of cells.8. Add 5 ml of cell suspension to each flask, cap the flasks, and return to the incubator.9. To determine when a medium change is required, check the culture every day for a drop in pH. This is usually evidence of depletion of medium by the growing culture. Examine cells under an inverted microscope for signs of cytoplasmic vacuolation, and estimate cell density.10. If only feeding is required, remove and discard medium.4.4 Viability measurementsViability is a measure of the proportion of live cells in a culture, as indicated by the ability of cells to divide or to perform normal metabolism. The most precise of all the methods of viability measurement is thecolony-forming assay. Here the ability of cells to grow is measured directly. A known number of cells at low density is allowed to attach and grow on the surface of a Petri dish. If the cell density is kept low each viable cell will divide and give rise to a colony or cluster of cells. From this, the plating efficiency’ is determined as the number of colonies scored per 100 cells plated X 100.1. Remove the cells from the attachment surface and suspend in solution.2. Add the cell suspension (cell concentration usually 100,200.400 per 35mm dish) to Petri dishes and incubate to allow colonies to form.3. Fix the colonies with methanol/acetic acid and slain with Giemsa staining.4. Count the number of colonies in each dish and calculate the average.4.5 Detection of bacteria and fungi in cell culturesMaterials and equipment:• Dehydrated media for bacterial/fungi cultivation.Steps1. Using an inverted microscope, equipped with phase contrast optics if possible, examine cell culture vessels individually. Cheek each culture first using low power. Batches of media should he tested for optimal growth promotion before use in cell culture quality control.2. After moving the culture to a suitable isolated area, remove aliquots of fluid from cultures that are suspect and retain these for further examination. Alternatively, autoclave and discard all such cultures.3. Prepare wet mounts using drops of the test fluids and observe under high power.4. Prepare smears, heat-fix and stain by any conventional method (e.g. Wright’s stain), and then examine under oil immersion.5. Consult for photomicrographs of representative contaminants and further details.6. Microscopic examination is only sufficient for detection of gross contamination and even some of these cannot he readily detected by simple observation. Therefore, an extensive series of culture tests is also required to provide reasonable assurance that cell lines stock or medium is free of fungi and bacteria.4.6 Fluorescent Procedure for Detecting MycoplasmaMaterials:1.Acetic acid/methanol: To make 90 ml, mix 30 nil of acetic acid with 60 ml of methanol.2.Hoechst 33258 stock (1 mg/ml): To make 10 ml. weigh 10mg of Hoechst 33258 and add 10 ml of HBSS. Store at 4℃in the dark.3. Hoechst 33258 working solution (100 ng/ml in HBSS): To make 50 ml, take 5ul of the Hoechst 33258 stock solution and add 50 ml of HBSS.4. Mounting mediumSteps:1.Plate cells on l2-mm glass coverslips placed in a 58-rnm tissue culture dish containing 8 ml of complete DMEM. Wait until the cells reach about 65% confluence.2. Rinse twice with NBSS.3. Add HBSS diluted 1:1 with Acetic acid/methanol (1:3). Rock the tissue culture dish and discard the solution4. Add acetic acid/methanol. Rock the tissue culture dish and discard the solution.5. Add acetic acid/methanol. Leave for 10 min at room temperature.6. Discard the acetic acid/methanol and wish twice with FIBSS.7. Add the Hoechst 33258 working solution (100 ng/rnL in If/IRS) and leave for IC mm at room temperature. ]he solution should be disposed in accordance with the safety regulations enforced in your laboratory.8. Rinse three times with HI3SS. Pick up the coverslip and place it cells slide down on a glass slide containing a drop of mounting medium. Leave for 1 hr at 37℃ or overnight at room temperature and observe in a fluorescence microscope using an appropriate filter. The Bisbenzamide fluorochrome excites at 360 nm and emits at 490-500nm. Cells free of mycoplasms should stain only in the nucleus. Contaminated cells exhibit bright, punctated staining in the cytoplasm.4.7 Procedure for Preserving and Storing Living CellsCryopreservatioflSteps:1.Select a 75-cm2 flask with cells in the logarithmic phase of growth and just approaching confluency. It may he necessary to renew the culture medium 24hr before harvesting the cells.2. Trypsinize the cells suspend the cells (pipette up and down) in complete DMEM at a concentration of2X106cells/ml. Check the cells for bacteria, fungal and mycoplasm.3. Centrifuge to pellet the cells and resuspend at 5-6X106 cells/ml in DMEM medium containing 10% FCS and 10% DMSO.4. Dispense the cell suspension in 1-mi aliquots into sterile cryoampoules. Tighten the screw cap and place in expanded polystyrene.5. Place the expanded polystyrene container with the ampoules at 4℃for l hr, and then place the container at -7012 or at -7012 for 15-20 mm then transfer to vapor-phase liquid nitrogen freezer.6. Note: Cells can he stored for many years. A temperature of 70℃is not adequate for storing cell lines except for very short periods (3-4 months). It is important to keep many ampoules in reserve. ThawingSteps:1. Remove the ampoules from the liquid nitrogen freezer, and immediately plunge the ampoule into water at 37℃until the liquid starts melting.2. Clean the ampoule all around with a tissue paper soaked with 70% ethanol,3. Add the contents of the ampoule to 5 ml of complete DMEM medium kept at 4℃. Centrifuge for 1-2 min at 300-400 g and resuspend the cells with 8 ml of complete DMEM at 37℃. Plate all the cells in a 25-cm2 flask and place it in the 37℃humidified 5% C02 incubator. Change the medium after 2hr when most of the cells have attached.4. Change the medium after 24 hr.5. Note: Caution should be exercised when removing ampoules kept in liquid nitrogen. Abrupt changes in temperature cause the ampoules to burst. Use safety glasses as well as insulated gloves. If the biological material is dangerous place it in a Cryoflex tubing (343958 Nunc) before freezing. Fetal Bovine Serum Lot Testing.4.8 Serum and medium testingBefore a new hatch of serum or media is purchased, ft is advisable to obtain a sample from the manufacturer and test its growth—supporting characteristics. Ibis is particularly important for serum. We usually select two of the most used cell types in the lab to test their growth and plating efficiencies.Steps:1 Make up individual aliquots of growth media, all containing the same media hatch, hut with the different test sera and including the serum batch currently being used.2 Plate the cells into 15 dishes for each test media at 5 / I o~ cells / 60 mm tissue-culture dish and containing 5ml of the media.3. Every day for 5 d thereafter, trypsinize the cells from triplicate plates and determine the cell numher/plate.4. Plot a growth curve (log cell number vs time), and calculated the doubling time and saturation density.5. At the same time as setting up the growth curves, seed in triplicate 60-mm dishes containing 5 ml of the appropriate media with 100 and 200 cells (6 plates/test).6. After 10-12 d fix the culture for IS mm by flooding with formalin.7. Tip the media and formalin (福尔马林) down the drain, and stain the clones with methylene (亚甲基) blue.8. Leave the stain for 15 min, and then wash it away with water9. Leave the plates stacked up against each other to dry in a 37℃room.10. Count the colonies.11. The three parameters of doubling time, saturation density, and plating efficiency should allow the section ofa serum that gives optimal growth.12. Note: This procedure need only be performed about every year. Enough serum can then be ordered for the next year, since the serum is stable at -20℃for at least 2 yr.4.9 Collagen isolation and coating of culture surface（第二次）Collagen types I can he obtain from the tendons that attach to the vertebrae of the rat tail.Steps:1. Cut fresh or thawed tails longitudinally from the base to the tip. Sterilized the tail with 70% ethanol.2. Pull the skin off the tail, remove the white collagen fibres and Sterilized with u.v. light.3. Stir 1 g of collagen fibres (from 3-4 tails) into 300 ml of 0.1% acetic acid for 48 h at 4℃.4. Centrifuge (4000 rpm, 30 mm 4℃), , obtain the upper liquid that is the stock solution of collagen.5.Dilute the stock solution 1:20 with double-distilled water,6. Cover the bottom of a plastic dish with this solution and air dry ( max, 37℃, 1-2 days).7. The dishes can be stored in a humid atmosphere at 4℃for several months, Alternatively, the stock solution may be neutralized and diluted in culture medium whereupon the collagen will gel due to the increased pH and salt concentration. Denature (air-dried) collagen enhances cell attachment (e.g. for endothelial cells and skeletal muscle), but native (undenatured) collagen gel may he required for correct phenotype expression.5. Disaggregation of the tissue and primary culture of Hen’s Egg（第三次课）5.1 Isolation of the tissueSteps:1. Incubate the eggs at 38.5℃in a humid atmosphere and turn through 180℃daily. Although hen’s eggs hatch at around 20 to 2ld, the lengths of the developmental stages are different from the mouse. For culture of。