Human gallbladder fibroblasts

1、 Basic cellular properties

Cell name:Human gallbladder fibroblasts

Organizational source: Gallbladder tissue

Species source: Human

Cell profile: Gallbladder,It is a pear shaped pouch structure located behind the liver below the right rib, which has the function of concentrating and storing bile. The gallbladder wall is composed of three layers: mucosa, muscle layer, and outer membrane. Among them, the outer membrane layer is mainly composed of fibroblasts.

Culture medium: primary fibroblast culture system

Fluid change frequencyChange the fluid every 2-3 days

Growth characteristics: Wall attached culture

Cell morphology: spindle shaped, irregular cells

Passage characteristics: Cell characteristics determine passage

Digestive fluid:0.25% pancreatic amylase

Cultivation conditions: Gas phase: Air, 95%; CO2 ，5%

2、 Usage:

Human gallbladder fibroblastsIt is a type of adherent culture of cells, with a long spindle shaped and irregular cell morphology. Under the standard operating procedures of the technical department, the characteristics of cells that can be passaged are determined for passage; We suggest that you conduct relevant experiments as soon as you receive the cells.

After receiving the cells, please proceed with the following steps for the customer.

1. Take out the T25 cell culture bottle, disinfect the bottle body with 75% alcohol, remove the sealing film, and place it in a cell culture incubator at 37 ℃, 5% CO2, and saturated humidity for 3-4 hours to stabilize the cell state.

2. Digestion of adherent cells

1) Suck out the culture medium from the T25 cell culture bottle and wash the cells once with PBS;

2) Add 1mL of 0.25% digestive solution to the T25 culture bottle, gently rotate the bottle until the digestive solution covers the entire bottom of the bottle, then aspirate excess digestive solution and incubate at 37 ℃ for 1-3 minutes; Observe under an inverted microscope, wait for the cells to shrink and become round, then add 5ml of culture medium to terminate digestion;

3) Gently blow and mix with a straw, inoculate T25 culture bottles according to the passage ratio for passage, then add fresh culture medium to 5mL, and incubate in a cell culture incubator at 37 ℃, 5% CO2, and saturated humidity;

4) After the cells adhere to the wall, culture and observe; Afterwards, replace the fresh culture medium according to the frequency of fluid exchange.

3. Cell detachment upon receipt

1) Collect all cell suspensions, centrifuge at 1000rpm for 5 minutes, and retain the precipitate;

2) Add 0.5mL of 0.25% digestive solution to a centrifuge tube, resuspend the precipitate, and let it digest at 37 ℃ for 3 minutes (or let it stand in a refrigerator at 4 ℃ for 5-7 minutes); After digestion, add 5ml of culture medium into the centrifuge tube to terminate digestion;

3) Centrifuge at 1000rpm for 5 minutes, discard the supernatant, resuspend the precipitate in 5ml of culture medium, and inoculate it into a new culture bottle;

4) After the cells adhere to the wall, culture and observe; Afterwards, replace the fresh culture medium (preheated at 37 ℃) according to the frequency of fluid exchange.

5) The adherent cells in the original bottle were digested normally.

4. Cell experiments

Due to the special nature of primary cell adhesion, when the adherent primary cells are transferred to other experimental vessels (such as glass slides, culture plates, confocal culture dishes, etc.) after digestion, the experimental vessels need to be wrapped to enhance cell adhesion and avoid cells from affecting the experiment due to poor adhesion; Mouse tail collagen is often used as a coating conditionI (2-5 μ g/cm2), Polylysine PLL (0.1mg/ml), Gelatin (0.1%), depending on the cell type. Suspended/semi suspended cells do not require encapsulation.

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Precautions for cell culture:

The company's products are only used for scientific researchPrecautions for primary cell culture. Primary cell culture is a very important experimental technique, mastering these precautions can make your experiment smoother.

Firstly, aseptic operation is crucial. Be sure to keep the operating environment clean and avoid bacterial or fungal contamination, otherwise the experiment will fail.

Secondly, choosing the appropriate culture medium and serum is also important. Different cells have different requirements for culture medium and serum, so the appropriate culture medium and serum should be selected according to the cell type.

Additionally, it is also a component in cell culture. Freshly prepared and regularly replenished during storage.

Static culture is also very important in the process of cell culture. After digestion and separation, cells need some time to adapt to the new environment, so it is important to avoid frequent removal of the culture bottle for observation during this period.

In addition, digestion time should also be controlled properly. Usually, it is sufficient to digest until visible small tissue particles are visible to the naked eye. Prolonged digestion time can lead to aggravated cell damage and affect the survival rate of cell culture.

Finally, for some special types of cells, it may be necessary to add some special growth factors to promote cell growth and proliferation. However, it should be noted that the cost of these growth factors is usually high.

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