Product Introduction
Cryopreserved Primary Tupaia Hepatocytes
(Plateable, Cat#LV-PTH001)
(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 tupaia hepatocytes are isolated from Tree Shrew or Tupaia belangeri in captivity, Using our company's independent intellectual property rights of efficient cryopreservation technology, standardized, large-scale isolation and cryopreserved primary hepatocytes can grow adherent after cell recovery, which can be used in hepatitis virus (HBV/HCV), new drug research and development and other fields. Cryopreserved primary tupaia hepatocytes are seeded into collagen-coated plates through the special recovery plating medium provided by our company, and the cells can be maintained for about two weeks after the cells are plated and replaced with maintenance medium. To preserve the original properties of the cells, it is recommended that hepatitis virus (HBV/HCV) infection and pharmacokinetic studies should be completed within 3 days of plating.
Ⅱ. Reagents and Materials
-Primary Tupaia hepatocytes (Cat#LV-PTH001)
-Recovery medium (Cat#LV-Rec001)
-Plating medium (Cat# LV-WEP003)
-Maintenance medium (Cat#LV-WEM003)
-Collagen-coated plate (Cat#LV-Coated)
-Sterile centrifuge tube of 15ml
-Wide-mouth pipette tip (Cut off the tip of normal pipette and sterilize it.)
-Centrifuge
-Pipette
-Thermostatic water bath
-Biosafety cabinet
-37 °C/5% CO2 incubator
Ⅲ. Recovery of Plateable Cells
1. In the biosafety cabinet, add 10ml of recovery medium (Cat#LV-Rec001) to a 15ml centrifuge tube and preheat in thermostatic water bath of 38 °C for 20 min. Preheat the plating medium in thermostatic water bath of 38 °C for 20 min.
2. Place the preheated recovery medium and plating medium in a biosafety cabinet. Rapidly transfer the frozen hepatocytes from the refrigerated location to a 38°C thermostatic water bath and immerse as much as possible in 38°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 amount 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 preheated recovery medium. (Note: there are more cells left on the cryopreservation tube and the pipette tip, aspirate 1ml recovery medium to rinse. Then, mix the 1ml recovery medium into the centrifuge tube medium). Slightly turn the tube upside down 2-3 times to mix them well.
6. Centrifuged 50×g of 15ml centrifuge tube at a low speed and at room temperature for 5 min. After the supernatant being removed, resuspend cells with 4ml plating medium.
7. Measure the viability and total number of living hepatocytes by Trypan Blue Exclusion Method. It is recommended to repeat 3 times to get an average. If the viability of cells is more than 70%, plate directly. Otherwise purify the hepatocytes before plating.
8. 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.
Ⅳ. Maintenance of Hepatocytes and HBV Infection
1. After culturing the hepatocytes for 6 hours, the cells have been well seeded. Remove the plating medium (containing part of the dead cells). Add the cells into plating medium and culture for 24h.
2. After 24h, add maintenance medium and change the medium every 2 days (Note: Never use PBS to clean cells).
3. Preparation of culturing infection medium: Pre-add HBV to the maintenance medium (HepAD38 MOI=500~1000, purified patient serum MOI=100~500, higher MOI may further improve the efficiency of infection).
4. Remove the old medium, add the infection medium, and incubate in a 37°C/5% CO2 incubator for 16h or overnight.
5. Remove the infection medium, rinse 3 times in PBS, add maintenance medium, and continue incubating in the 37°C/5% CO2 incubator
6. Collect the supernatant every 2 days after HBV infection for viral antigen and HBV DNA detection. Collect 3dpi, 5dpi, 7dpi, 9dpi supernatant. 7dpi is to end the cell culture, longer culture time depends on cell status. (dpi: days of culturing after the infection)
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. 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.
Ⅵ. Contact Information
Tel:0755-28284050
Technical Support: 19902901483 (Dr. Zhou)