Product Introduction
Catalog:LV-HSC001
Number of cells:1 million/vial
Form:cryopreservation (spot/P1 generation), fresh culture (need to be customized)
Quotation:Please contact (19902901483)
Uses: For research use only
Human hepatic stellate cells
(Can be attachment,Cat# LV-HSC001)
(For research use only)
The instruction is suitable for the human hepatic stellate cell of our company. Please follow the instructions,when you use it. If you have any questions, please consult the technicians.
I Introduction
Liver stellate cells (HSCs) make up about 30% of non-parenchymal cells. HSCs are present in the Disse cavity, with fusiform or polygonal. There are multiple vitamin A-rich droplets in the cytoplasm, and their elongated protrusions extend outward around the endothelial cells of the blood sinuses, which is the primary site of storage of retinal derivatives in the body. In normal liver, stellate cells are quiescent, do not express α smooth muscle actin (α-SMA), have low proliferative activity, and have low ability to synthesize collagen. Its main function is to store retinal. This product uses enzyme perfusion digestion to isolate donated material from the human liver, retaining the biological activity of cells in the body. Other liver cells are removed by density gradient centrifugation with ultra-high purity (>85%). All cells were tested to be free of bacteria, fungi, actinomycetes and other contamination, and the cell viability was high (cryopreservation >85%,fresh >95%,cell viability after isolation > 95%). The company can personalize the primary cells to meet the needs of different customers.
II Reagents and Materials
- Hepatic stellate cells(Cat# LV-HSC001)
- Recovery medium(Cat#LV-Rec001)
- Stellate cell culture medium (Cat# LV-HSCM001)
-Crushed ice and ice boxes
- Sterile centrifuge tube of 15 ml
- Disposable pipette
- Wide-mouth pipet tip (The tip of a normal one is cut off and sterilized)
- Pipette
-Thermostat water bath (Preheat at 38℃)
- Refrigerated horizontal centrifuge (With horizontal rotor,it is possible to centrifuge 15 ml of centrifuge tubes)
- Biosafety cabinet
- 37 °C/5% CO2 incubator
- 75% alcohol
III Resurgence and Plating of Cells
1. Insert the 15 ml centrifuge tube into an ice box containing enough crushed ice and sterilize it UV for 15 min. Precool centrifuge.
2. The recovery medium should be placed in crushed ice for sufficiently precooling, and the plating medium should be placed in a 38 °C thermostat water bath to be fully preheated.
3. Quickly transfer the frozen hepatic stellate cells from the refrigerated position to a thermostat water bath of 38℃. Then,immerse them in as much water as possible at 38°C and shake clockwise. Please make sure that the cap of the freezing tube is kept above the water.
4. Thaw the freezing tube for about 90-120s, until only a small amount of crushed ice floats in it.
5. Sterilize the freezing tube with 75% alcohol and transfer it to a biosafety cabinet.
6. Resuspend the cells with a wide-mouth pipet tip (gently blow 2 times) and transfer to a pre-cooled centrifuge tube of 15 ml.(Note: if there are more cells left on the freezing tube and the pipette tip, aspirate 1 ml of resuscitation medium to clean the them.
7. Add prechilled recovery medium to the cell suspension dropwise and add 10 ml of recovery medium to every 1 ml of cell suspension (Note: the first 3 ml should be added dropwise and shaken slightly, and the next 7 ml can be accelerated). Finally, slightly upside down 1 time to mix well.
8. Centrifuge of 600 × g at 4 °C for 5 min, remove the supernatant and resuspend with plating medium.
9. The pelleted cells should be resuspended with hepatic stellate cell culture medium and set the volume to 4 ml. The viability and total amount of hepatic stellate cell can be determined by trypan blue exclusion.
10. Cells should be seeded into a Petri dish at the density of 3×104cells/cm2, shaken well and cultured in a 37 °C/5% CO2 incubator. Change the solution after 24h.
IV Culture and Passage of Cell
1. The cells can be passaged when the degree of fusion reaches 80%.
2. Put the medium, PBS and pancreatin into thermostat water bath of 37℃ for preheating. And wipe it with 75% alcohol before placing in the ultra-clean table.
3. Aspirate the old culture solution and add a small amount of PBS to wash the cells. In addition, add an appropriate amount of pancreatic enzyme so that the amount of pancreatic enzyme can cover the cells. Incubate the cells at 37°C for 2-3 min and observe them under the microscope.
4. Discard the pancreatic enzyme when the intercellular space of the adherent cell becomes larger and the cells tend to be rounded but have not yet floated. After that, fresh culture medium should be added and the cell flask should be shaken to terminate the action of pancreatin. Then, carefully blow the adherent cells with a pipette to make a cell suspension (Note:Pay attention to the force of the blowing to avoid generating a large number of bubbles).
5. Centrifuge of 600 × g at 4 °C for 5 min, remove the supernatant and resuspend with the culture medium.
6. Inoculate the cell suspension into a new flask/plate at the density of 3×104cells/cm2 and place it in a 37 °C/5% CO2 incubator to observe the adherent growth every other day.
V Customer Service
If you find any quality problems with the product, please collect the original data and contact the company's salesmen or technical support at the first time. The company ensure after-sales service. Every laboratory has different conditions, different operating habits, different proficiency, and objective factors in experimental failure. If the operation is not strictly in accordance with the instructions or exceeds the time limit of after-sales, the company does not do after-sales. Please understand and support us.
Validity period and raw data provided:
Resuscitation problems: Within 24 hours of resuscitation, trypan blue staining or PI staining should be provided.
Pollution problems: Within 96 hours of resuscitation, microscope photographs of differences should be provided.
Purity issues: Within one month, immunofluorescence or flow cytometry results should be provided
VI Contact Number
Tel:0755-28284050
Marketing representative: 18129812531 (Manager Deng)
Technical Support: 19902901483 (Dr. Zhou)