What issues need to be paid attention to in cell culture

1. The experimental materials should be fresh, and the materials should be stored at low temperature after being separated from the living body, and the cell separation experiment should be carried out as soon as possible.

2. Aseptic operation. The culture medium used during the operation can be added with 5 times the usual content of penictomycin in the cell culture medium.

3. When separating cells by enzymatic method, pay attention to the concentration of the enzyme solution and control the digestion time.

When separating cells by patch method, pay attention to the gentle action and do not hurt the cell tissue, and the edge of the tissue block should be as flat as possible to facilitate the cell free.

4. Selection of culture medium. Different cells have different requirements for nutrients in the culture medium, which are selected according to the characteristics of the isolated cells.

Subculture cell culture:

1. Aseptic operation.

2. Control the digestion time.

3. Pay attention to the growth status of cells in time.

1. To ensure the freshness of the experimental materials, pay attention to low temperature preservation after separating the materials from the living body.

2. Aseptic operation should be ensured. It is possible to add 5 times the usual amount of penictomycin to the culture medium used during operation, which is not pure.

3. Pay attention to the concentration of the enzyme solution and control the digestion time.

4. Pay attention to the selection of culture medium. Different cells have different requirements for nutrients in the culture medium, and are selected according to the characteristics of the isolated cells.

Aseptic operation, control of digestion time, timely attention to the growth state of cells.

Recently, the author has been culturing human umbilical vein endothelial cells (HUVECs) to record some basic operations of Zhiqiao in cell culture.

Take out the cell culture flask from the incubator with 5% CO2 at 37°C, first observe whether the liquid in the flask is cloudy (turbidity indicates that the cells may be contaminated), then observe the growth of the cells under the microscope, and when the cells are more than 70% long, they can be passaged.

Add 3ml PBS to the 25ml culture flask and wash it twice, then add trypsin (cactus pancreatic enzyme used in the laboratory is more warm and silvery), put it in the incubator and digest for 3-5min (to ensure that the cells are digested), and add 2ml of culture medium (ECM) to stop the digestion. Collect the liquid into a centrifuge tube, centrifuge at 350g for 5min (so that the cells can be precipitated), then remove the supernatant, add 6ml ECM to the centrifuge tube, blow the cells well (blow into single cells), add 2ml to each of the three 25ml flasks, and then add the medium to 6ml in the flask.

After the cells in the 25 ml flask are full, wash twice with 3 ml PBS, centrifuge after trypsinization, discard the supernatant, add medium and 500 L DMSO (10%), blow the cells well, and aliquot into 5 cryovils, 1 ml each, and put in -80 cryopreservation.

After the cryopreserved cells are taken out from -80 or liquid nitrogen, immediately put them in warm water at 37 °C (pay attention to whether liquid nitrogen leaks into the cryopreservation tube), after thawing, centrifuge at 350g for 5min, discard the supernatant, add 1ml ECM, blow the cells well, add 25ml culture flask, and make up ECM to 6ml.

Note: a. The lid of the culture flask should be passed through the alcohol lamp, and the cap of the culture flask should not be tightened;

Two. The 75ml flask is a 10ml culture system, plus trypsinization, and 4 times the amount of medium to terminate digestion; The 6-well plate is a culture system (trypsinized), the 12-well plate is a 2 ml culture system (trypsinized), the 24-well plate is a 1 ml culture system (trypsinized), and the 96-well plate is a 100 L culture system (1 drop trypsinized). After adding trypsin, shake the flask or culture plate slightly so that the trypsin can cover the adherent cells;

Three. The amount of PBS used for cleaning is half the amount of medium;

Four. The 25 ml flask is full of cells and there are about one cell;

Five. Huvec is no longer available after the 4th generation.

Brineco Biotechnology - Answers for you: 1. The right cell culture mediumThe right cell culture medium is one of the most important conditions for cell growth and proliferation in vitro. The culture base not only provides cell nutrition and the basic material to promote cell growth and proliferation, but also provides a living environment for the growth and reproduction of cultured cells.

2. High-quality serumAt present, most synthetic media need to add serum. Serum is one of the most important components of cell culture media, containing a variety of growth factors and other nutrients required for cell growth. 3. Sterile and non-toxic cell culture environment: Sterile and non-toxic operation environment and culture environment are the primary conditions to ensure the success of cell culture in vitro.

Due to the lack of defense against microorganisms and toxic substances, cells cultured in vitro can lead to cell poisoning and death once they are contaminated by microorganisms or toxic substances or accumulate their own metabolites. Therefore, when culturing cells in vitro, it is necessary to keep the cell living environment sterile, non-toxic, and remove cell metabolites in time. 4. Constant cell growth temperature: To maintain the vigorous growth of cultured cells, there must be a constant and appropriate temperature.

5. Suitable gas environment gas is one of the necessary conditions for the survival of mammalian cell culture. The main gases required are oxygen and carbon dioxide.

1. Laboratory design.

Cell culture is an aseptic technique that requires a working environment and conditions that are free of microbial contamination and other harmful factors. Cell culture chambers and design principles are designed to prevent microbial contamination and harmful influences, and require a clean, fresh, dry and smoke-free working environment. Cell culture work includes:

Preparation of working solution, aseptic operation (sampling), incubation, aseptic treatment, storage of cells and supplies, etc. The design and implementation principle of the cell culture room is generally that the aseptic operation area is located on the inner side of the room with less movement, and the routine operation and closed culture are in one room, and the scrubbing and disinfection are in the other room.

2. Commonly used facilities and equipment.

1) Ultra-clean workbench: also known as purification workbench, it is divided into three categories: side-flow type, DC type and external flow type.

2) Aseptic operation room: It is generally composed of three parts: changing room, buffer room and operation room. The operation room is equipped with a purification bench, a carbon dioxide incubator, a centrifuge, an inverted microscope, etc. The buffer room can be used to store refrigerators, refrigerators, and sterilized sterile items.

3) Operation room: ordinary incubator, centrifuge, water bath, timer, ordinary balance and daily analysis and processing items.

4) Scrubbing and disinfection room: oven, sterilizer, distilled water processor and acid tank, etc.

5) Analysis room: microscope, computer and printer, etc.

3. Culture vessels.

Cell culture is dominated by glassware, and three times the maximum amount required is often prepared. Utensils should be made of glass products with good transparency, non-toxicity and neutral hardness. The following types of glassware are commonly used.

1) Liquid storage bottle: used to store various prepared culture fluids, serum and other liquids, often replaced by 500ml, 250ml, 100ml normal saline bottles or plasma bottles.

2) Culture flask: according to the type of cultured cells, the shape of different culture flasks are different, the cells used for cell subculture require uniform wall thickness, which is convenient for cell adherent growth and observation, the size of the bottle mouth should be consistent, the caliber is generally not less than 1cm, and the straw is allowed to extend into any part of the bottle, and the specifications are 200ml, 100ml, 50ml, 25ml, 10ml and so on. A commonly used 10ml ordinary round flask for peripheral blood culture.

Both flasks require to be made of high-quality glass.

3) Petri dish: used for open culture and other purposes. It is divided into diameters of 30mm, 60mm, 120mm, etc.

4) Straws: There are two types of straws: long straws and short straws, and long straws are also called graduated straws. The upper part of the modified tube has a spherical scale called the modified straw, and the graduated straw is used to move the liquid.

There are two commonly used types: 1ml and 10ml. Short straws, also called droppers, are divided into two types: elbow and straight.

5) Centrifuge tube: centrifuge tube is the most widely used vessel in cell culture, according to the use of different forms, commonly used in cell culture centrifuge tube has two types: pot-bellied culet centrifuge tube and ordinary cuvette centrifuge tube. The former are 50ml, 30ml and 15ml respectively; The latter are mostly 10ml and 5ml.

6) Others: such as triangular flasks, beakers, graduated cylinders, funnels, syringes, etc.

For cell recovery, we can recommend BioLife's Thawstar series cell program resuscitation device.

BioLife BioLite Thawstar Cell Program Resuscitation Apparatus.

The storage and transport of cells should be realized under cryopreservation conditions, and the extraction and application of cells usually use the thawing and resuscitation method of water bath, while the traditional thawing and resuscitation of water bath is faced with the inability to control and manage, and the subjective misjudgment of human beings and the contamination of water bath bring certain risks.

In April 2019, Biolife Solutions wholly acquired the intellectual property rights of Astero and its cell program resuscitation company, and the development of high-end models of THAWSTAR cell program resuscitation technology has been funded, among which CFT series models have been used in biopharmaceutical companies, R&D companies, university research units, and even hospital laboratories around the world, and the recovery effect has been recognized by the industry. As a partner of Astero China, Shanghai Laxi Industrial Technology Co., Ltd. sells a full range of Astero products in Chinese mainland, Hong Kong and Taiwan.

Thawstar Cell Program Resuscitation Apparatus.

It is an instrument for the programmed recovery of cells. Compared with traditional water bath operation, the instrument is simple to operate and has good repeatability, which can effectively avoid the risk of contamination during operation. Since the instrument automates a series of procedural resuscitation operations, the risk of incorrect operation and contamination based on the subjective guess of the operator is eliminated and the reproducibility is high!

The THAWSTAR system is suitable for frozen cells preserved on dry ice, -80 freezers, liquid nitrogen, etc. The operation is simple, for example, the ThAWSTAR CFT2 only needs to be inserted into the vial and wait for it to finish, and when the processing is complete, the vial will be automatically ejected.

The THAWSTAR CFT Cell Program Resuscitation Device can be performed directly in the BSC of the Biosafety Cabinet!

1. Recovery.

1.Remove the vial from the liquid nitrogen and immediately place it in a 37 water bath with slight shaking. After the liquid has melted (about a few minutes), take it out and spray some alcohol and put it in the clean workbench.

2.Aspirate the above cell suspension into a 15 ml centrifuge tube containing 10 ml of culture medium (wash the cryovial with the medium to wash off all the cells stuck to the wall) and centrifuge at 1000 rpm for 5 minutes.

3.Pour off the supernatant and add 1 ml of medium to suspend the cells. Aspirate into a 10 cm dish filled with 10 ml of medium and gently shake it back and forth, left and right, so that the cells in the dish are evenly distributed.

4.Mark the cell type and date, the name of the culturer, etc., put it in the CO2 incubator for culture, and change the medium after the cells are adherent.

5.Change the medium every 3 days.

2. Generation.

1.Passage when the cell coverage in the dish reaches 80%-90%.

2.Aspirate the original medium.

3.Add appropriate trypsin (as long as it can cover the cells) and digest for 1-2 minutes.

4.After the cells are rounded, add an equal volume of serum-containing medium to terminate digestion.

5.Pipette the cells to suspend them.

6.Aspirate the cells into a 15 ml centrifuge tube and centrifuge at 1,000 rpm for 5 min.

7.Pour off the supernatant, add 1-2 ml of medium, and blow the cells all up.

8.Depending on the cell type, the cells are transferred to several dishes. In general, there are 5 cancer cells and 3 normal cells. Continue to cultivate.

3. Cryopreservation.

Digest the cells and centrifuge (ibid.). Suspend the cells with the prepared cryopreservation solution, aliquot them into sterilized cryopreservation tubes, let them stand for a few minutes, and indicate the cell type and cryopreservation date. 4 30min, -20 30min, -80 overnight, and then put into liquid nitrogen irrigation for storage.

Preparation of cryopreservation solution: 70% complete medium + 20% FBS + 10% DMSODMSO should be added slowly and shaken as you go.

Pay attention to aseptic operation!