Animal cell and embryo engineering experiments commonly used culture solution preparation and detection experiments
Animal cell and embryo engineering experiments commonly used culture solution preparation and detection experiments
Common culture solution preparation and testing for animal cell and embryo engineering experiments for (1) animal cell culture (2) embryo engineering development (3) molecular biology research.
Operation method
Solution Preparation Method
Principle
The main objects of animal cell engineering are tissues, organs or cells of animal organisms under isolated conditions, and these tissues or organs used for isolated culture are called explants; the main objects of animal embryo engineering are gametes and embryos. In order to satisfy the normal survival and growth and development of the operation object under the conditions of isolation, it is necessary to provide it with good nutritional conditions suitable for growth and development, i.e., culture fluid conditions. There are many differences between animal tissues, cells or gametes and embryos in terms of nutritional metabolism, growth and development, and the nutritional requirements are not the same. Therefore, when performing in vitro manipulation or culture of animal tissues, cells and gametes, embryos, different manipulation fluids or culture fluids need to be prepared. Therefore, understanding the composition of culture fluid and mastering its preparation method is a basic task in cell and embryo engineering research and experiments. Due to the characteristics of animal cell and embryo engineering operation objects, the commonly used operation fluids and culture fluids are usually prepared in liquid state. Some components in the operating fluid and culture fluid are unstable in the solution, so the most basic components of the liquid are firstly allocated into the base fluid, and then these components are added to the base fluid as additives to form the operating fluid and culture fluid before use, and the way of adding these components varies among different culture fluids and different laboratories. The operation of animal cell and embryo engineering is based on aseptic conditions, which requires the establishment of a set of aseptic operation techniques and aseptic culture environment, and the preparation of aseptic liquid is the most basic condition. The liquid often carries various kinds of stray bacteria in the process of preparation, and should be sterilized immediately after the liquid is prepared, or the sterilization process should be completed within 24 hours. Since the operating fluid and culture fluid of animal cell and embryo engineering often contain substances that are easy to decompose when heat is encountered in the composition, so the filtration method is mostly used to remove the bacteria in the operating fluid and culture fluid, and the filtration and sterilization is completed when the dispensing is completed. In order to check whether the prepared liquid is still contaminated with bacteria, the prepared liquid should be examined at certain time intervals, such as whether the color changes, whether turbidity occurs, and whether there are colonies, etc., and if necessary, it can be cultured and examined.
Materials and Instruments
Alcohol Distilled water NaOH HCl Move I. Preparation of liquid preparation Caveat 1. The culture of animal cells needs to be carried out in a strictly sterile environment. 2. Amino acid is the basic unit of protein, different cells need different kinds of amino acids, according to different cultured cells, add different essential amino acids in the culture solution. Common Problems 1. Primary cells: all kinds of animal tissues are taken out from the organism, processed by various enzymes (commonly used trypsin), chelating agents (commonly used EDTA) or mechanical methods, dispersed into single cells, and then cultured in the appropriate medium to enable the cells to survive, grow and reproduce, this process is called primary culture. 2. Passage cells: the basis of all cell biology experiments. When the cells are full grown in the culture flasks, they need to be diluted and seeded into multiple flasks in order for the cells to continue to grow, this process is called passaging. Passage culture can obtain a large number of cells for experimental needs. 3. Animal cell culture medium is divided into synthetic medium and natural medium according to its source (the majority of the currently used medium is synthetic medium), according to its material state is divided into dry powder medium and liquid medium two categories. For more product details, please visit Aladdin Scientific website.
Ultra-clean workbench, alcohol cotton ball alcohol spray bottle detergent Bucket bottle brush test tube brush ultrasonic cleaning instrument water control rack drying box electric furnace autoclave sterilizer blower drying box gauze cotton cloth kraft paper sulfuric acid paper cotton rope aluminum box alcohol lamp alcohol cotton ball precision electronic balance medicine spoon weighing paper beaker glass rod absorbent paper dropper bottle dropper tube measuring cylinder syringe pipette funnel volumetric flask magnetic stirrer acid meter pH test paper needle filter filter film penicillin vials Serum bottles Bottle stoppers Sealing film
1. Inspection and commissioning of instruments and equipment
Liquid preparation involves instruments and equipment, such as autoclave sterilizer, blast drying oven, precision electronic balance, ultra-clean bench, etc., in accordance with the instructions before use, in the working state of the inspection, debugging.
2. Washing and sterilization
In accordance with the cleaning procedures described in Part II, the items in contact with the liquid and its components are cleaned, rinsed with distilled water and dried at 60 ℃. Used for drug reagent dissolution, mixing, volume setting of the vessel can be used. Used for aseptic treatment of liquids and dispensing of vessels and articles for packaging, high-temperature-resistant vessels and articles using constant-temperature blast drying oven for dry heat sterilization; not high-temperature-resistant articles using autoclave steam sterilizer for wet heat sterilization, wet heat sterilization of the articles should be re-drying.
II. Preparation of common operating fluid and culture fluid
Take the commonly used operating fluid and culture fluid in the experimental guidance program as an example to introduce the liquid preparation method.
1. Ca2+-free, Mg2+-free phosphate buffer solution [PBS (-)
Ca2+-free, Mg2+-free phosphate buffer solution (phosphate buffer solution, PBS) is commonly used as cell culture operating fluid. The preparation method is as follows: weighing NaCl 10.0 g, KCl 0.25 g, Na2HPO4-12H2O 1.44 g, KH2PO4 0.25 g, 500 mL of tetradistilled water, fully dissolved with magnetic stirring in a beaker, 1000 mL volumetric flask, dispensing, autoclaved at 15 lb for 30 min, and stored in a refrigerator at 4 ℃.
2. 0.25% trypsin cell digestion solution
Trypsin 0.25 g, EDTA 0.04 g, PBS (-) solution 100 mL, magnetic stirring in the beaker to fully dissolve, 0.22 um filter membrane positive pressure filtration, 4mL/bottle dispensing, -20 ℃ refrigerator storage.
3. DMEM cell culture medium
DMEM liquid (Dulbicco's minimal essential medium) was mainly used for cell culture. Weighing high sugar DMEM powder 13.4 g, NaHCO3 3.7 g, respectively, add four distilled water 300 mL, magnetic stirring to fully dissolve, and then NaHCO3 solution slowly added to the DMEM solution, 1000 mL volumetric flask was fixed, adjusted pH7.2, added 110 mL serum, 80 units / mL penicillin, 100 units / mL streptomycin, 0.22 um membrane positive pressure filtered, partitioned, and stored in 4 ℃ refrigerator.
4. Modified Duchenne phosphate buffer solution (mPBS)
Modified phoaphate buffer solution (mPBS) is often used as the operation solution for embryo recovery (egg flushing solution). 
Preparation process:
(1) Prepare A, B, C three solutions respectively, the amount of three distilled water added to 80% of the final amount, placed in the refrigerator freezer to cool down and then mixed sequentially, which can prevent precipitation, and then used a volumetric flask was fixed to 1,000 mL. In order to indicate the pH value, phenol red can be added, but it is not a component of the PBS, and 1.0% ( g-mL-1 ) solution 1.0 mL-L-1 is generally added.
(2) with 0.2 mol-L-1NaOH or 0.2 mol-L-1HCl to adjust the pH to 7.1 ~ 7.2 (such as distilled water is fresh, weighing is accurate, formulated after the PH that is 7.1 ~ 7.2, do not have to adjust. In practice, if the pH is not allowed, it may be a problem of distilled water, reagents or weighing accuracy). Make the osmolality stable at about 290 millimolar osmolality concentration (mOsm).
(3) If the reagent contains water of crystallization that does not match the formulation, it should be converted to molecular weight. For reagents susceptible to moisture absorption, bake and dry prior to formulation. The drying temperature should be consulted in the chemistry manual, in order not to decompose the reagent or lose the water of crystallization in the molecule.
(4) Remove bacteria by suction filtration with a G6 filter, or by positive pressure filtration after installing a filter membrane with a needle filter.
(5) Strictly observe the aseptic operation procedures in the preparation process, especially in filtration and dispensing. The prepared liquid is divided and sealed, labeled with the date of preparation. 4 ℃ refrigerator can be stored for 1 ~ 4 months. It should not be stored in refrigerator freezer or low-temperature refrigerator, otherwise salt crystals will be precipitated.
(6) Add bovine serum albumin or inactivated fetal calf serum or newborn calf serum as needed before use. Generally, the concentration of serum added to the egg wash is 2% to 5%. For the convenience of use, serum can be added before filtration, and then filtered and dispensed. Serum inactivation can remove the toxic embryonic factors in serum, inactivation method is to maintain the serum at 56 ℃ for 3O minutes.
5. CZB liquid
CZB liquid is a culture medium published by Chatot CL, Ziomek CA and Bavister BD in 1989, which is specially designed to overcome the 2-cell block in the in vitro culture of mouse embryos, and is abbreviated as CZB liquid by the abbreviation of the names of the three persons. Its basic components are shown in Table 4-2. It is characterized by the use of glutamine to overcome 2-cell block in mouse embryos without the use of glucose, which may lead to delayed embryonic development and developmental block, at the early oogenesis stage. However, early embryonic development to the late stage requires 48 hours of incubation in CZB fluid containing a glucose concentration of 5.56 mM, which can well support the development of mulberry embryos to blastocysts. In recent years, CZB fluid and its modified fluids are often used as fluids for in vitro manipulation and culture of mouse embryos.
6. KSOM Liquid
KSOM fluid was first published as SOM fluid (Simplex Optimized Medium) by Lawitts and Biggers (1991,1992), which can support fertilized eggs to overcome the 2-cell block. Later, after the NaCl and KCl concentrations of the SOM fluid were increased (Lawitts and Biggers 1991, Erbach et al 1994), the new fluid became known as KSOM. kSOM fluid has a high rate of cell division and a high rate of blastocyst development when cultured in mouse embryos. the basic components of KSOM fluid are shown in Table 4-3. 
Preparation method of CZB liquid and KSOM liquid: dissolve basic components such as NaCl, KCl, KH2PO4, NaHCO3, sodium lactate and additive components such as sodium pyruvate, EDTA, penicillin and streptomycin with tetra-distilled water or ultra-pure water, which together make up Liquid A. CaCl2 and MgSO4. 7H2O were dissolved as Liquid B and C, respectively. The three liquids were mixed after cooling down at 4 ℃ refrigerator to prevent precipitation, and the mixture was fixed with distilled water. After that, phenol red (1%, mL-mL-1, addition volume 1000 mL-L-1 ) was added, the color was observed and the pH was determined with precision test paper and instruments, and the pH was adjusted to 7.3 ± 0.1 with 0.2 mol-L-1 HCl or 0.2 mol-L-1 NaOH. Bacterial filtration was carried out with a filter and divided into equipments for use. Glutamine (final concentration is generally 1 mmol-L-1, master batch 1%, add 10 mL per mL), glucose is first formulated into a high-concentration master batch, and then add the master batch into the culture solution to reach the final concentration; serum or BSA is divided into small packages, and then added into the culture solution at the time of clinical use, and the final concentration of serum is generally 10-20% (V/V), and the final concentration of BSA is usually 4000 mg-L-1. The final concentration of serum is usually 10-20% (V/V), and the final concentration of BSA is usually 4000 mg-L-1. After adding serum, BSA, and glutamine to the culture solution at the desired concentration, the bacteria are filtered again using a 0.22 um filter membrane. In addition, it should be noted that when adding BSA to the culture medium, do not stir or shake vigorously to avoid producing a large number of bubbles, and the required amount of BSA should be sprinkled on the surface of the liquid to be dissolved by autolysis or gently shaken to dissolve.