Product Introduction
Product |
Primary Human Hepatocytes (Technical Service) |
Catalog |
LV-PHH001 |
Specification |
4-8 million |
Strains |
Chinese |
Number of donors |
N=1 |
Free shipping or not |
No |
Delivery |
1 week |
Carriage |
Liquid nitrogen |
Storage |
Liquid nitrogen |
Life cycle |
10 years |
Warranty |
7 years |
|
Primary Human Hepatocytes
(For research use only)
In order to ensure the safety and biosafety of the experimenters, please wear necessary protective equipment such as protective masks, latex gloves and protective eye shields (during revival) when contacting this product and its waste. Please operate in strict accordance with this manual, and the waste generated after the experiment must be harmless treated in accordance with the relevant laws and regulations to ensure biological safety.
Ⅰ. Introduction
Liver-Biotech is a national high-tech enterprise focusing on the isolation, cryopreservation and regeneration of primary hepatocytes. Our primary human hepatocytes are isolated from the human liver tissue obtained from regular hospitals. All the materials are clearly sourced and the patients or their family members have given informed consent. After a variety of tests, the recovery cells are highly viable and polarized (differentiated), and could be widely used in basic life science research and drug discovery.
II. Reagents and Materials
-Primary human hepatocytes
-Recovery medium (Cat#LV-Rec001/3)
- Purification medium (provide if needed)
-Plating medium
(Plateable cells are needed. Cat#LV-WEP001)
-Maintenance medium
(Plateable cells are needed. Cat#LV-WEM001)
-Collagen-coated plate
(Plateable cells are needed. Cat #LV-Coated)
- Sterile centrifuge tube of 15ml
- Wide-mouth pipette tip (Cut off the tip of normal pipette and sterilize it.)
- Pipette
-Thermostatic water bath (Preheat at 37℃. Please calibrate the temperature with a thermometer)
- Low-temperature horizontal centrifuge (with a horizontal rotor, it is possible to centrifuge a 15ml centrifuge tube)
- Biosafety cabinet
- 37 °C/5% CO2 incubator
III. Resurgence of Suspension Cells
1. Add 10ml recovery medium (Cat#LV-Rec003) to a 15ml centrifuge tube in a biosafety cabinet, pre-warm a
thermostatic water bath at 37°C for 20 min, and then transfer the medium to a biosafety cabinet, and pre-warm the plating medium at 37°C.
2. Rapidly transfer the frozen hepatocytes by a liquid-nitrogen thermos from the refrigerated location to a 37°C thermostatic water bath and immerse as much as possible in 37°C water with clockwise horizontal rolling, but must ensure that the cap of cryopreservation tube stays above the water.
3. Thaw the cryopreservation tube for about 90 ~120s until only a small piece of crushed ice floats in it.
4. Sterilize the cryopreservation tube with 75% alcohol and transfer it to a biosafety cabinet.
5. Aspirate the cells with a wide-mouth pipette tip and add them dropwise to a 15ml centrifuge tube containing the preheated recovery medium. (Note: there are some cells left on the cryopreservation tube and the pipette tip, aspirate 1ml recovery medium to rinse. Then, transfer the 1ml recovery medium into the 15ml centrifuge tube). Slightly turn the 15ml centrifuge tube upside down 2-3 times to mix cells well.
6. Centrifuge 50×g of the 15 ml centrifuge tube at low speed (Higher centrifugation speed may has an adverse effect on cell viability), at room temperature for 5min. After the supernatant being removed, resuspend cells with a metabolism medium (provided by the customer) for drug metabolism, and the viability and number of viable hepatocytes were determined by Trypan Blue Exclusion Method (manual counting on a hemocytometer, not recommend the cell counter machine). Add the drug according to the experimental requirements and measure the metabolites.
Ⅳ. Recovery of Plateable Cells
10. Seed the living cells at the density of 1.5-1.8×105cells/cm2 onto collagen-coated plate. Shake the plate well and incubate it in a 37℃/5% CO2 incubator for 24h.
Ⅴ. Maintenance of Hepatocytes and HBV Infection
1. After culturing the hepatocytes for 24 hours, the cells have been well seeded. Remove the plating medium (containing part of the dead cells), add the maintenance medium, and change the culture medium every 2 days.
2. Preparation of culturing infection medium: Add 4% PEG8000 to the maintenance medium and mix well; Hepatitis B virus (HepAD38 MOI=500~1000, purified patient serum MOI=100~500, higher MOI may further increase infection efficiency).
3. Remove the old medium, add the infection medium, and incubate in a 37°C/5% CO2 incubator for 16h or overnight.
4. Remove the infection medium, rinse 3 times in PBS, add maintenance medium, and continue incubating in the 37°C/5% CO2 incubator.
5. Collect the supernatant every 2 days after HBV infection for viral antigen and HBV DNA detection. Collect 3dpi, 5dpi, 7dpi, 9dpi supernatant. 9dpi is to end the cell culture, longer culture time depends on cell status. (dpi: days of culturing after the infection).
Ⅵ. 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. We ensure your after-sales service. There are some objective factors for the failure of the experiment, for example, different laboratory environments, different operating habits and operators’ proficiency, etc. If the operation is not strictly in accordance with the instructions or exceeds the time limit of after-sales service, our company will not provide you with after-sales service. Thank you for your understanding and support.
Validity period of after-sales and original data needed:
Recovery problems: Trypan Blue staining or PI staining should be provided within 24 hours of recovery.
Pollution problems: microscope photographs of differences should be provided within 96 hours of recovery.
Purity problems: immunofluorescence or flow cytometry results should be provided within one month.
Ⅶ. Contact Information
Tel:0755-28284050
Technical Support: 19902901483 (Dr. Zhou)